Segmented Polymers and Their Conjugates

ABSTRACT

Segmented water soluble polymers, containing a higher molecular weight segment linked to a lower molecular weight segment, are described. In one embodiment, the polymer segments are poly(ethylene glycol) segments. The segmented polymers are functionalized and are useful for conjugation to various moieties such as pharmacologically active substances. Also described are conjugates of such polymers and methods of their preparation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/402,350, filedApr. 11, 2006, which is a divisional of U.S. Ser. No. 10/734,858, filedDec. 11, 2003, now U.S. Pat. No. 7,053,150, which is acontinuation-in-part of U.S. Ser. No. 10/024,357, filed Dec. 18, 2001,now U.S. Pat. No. 6,774,180, which claims the benefit of provisionalapplication U.S. Ser. No. 60/256,801, filed Dec. 18, 2000, all of whichare hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to medium to high molecular weight,functionalized, water soluble polymers useful for conjugation to variousmoieties, including pharmacologically active substances. In particular,the invention relates to functionalized water soluble polymers, such aspoly(ethylene glycols), containing a higher molecular weight segmentlinked to a lower molecular weight segment, and methods of theirpreparation.

REFERENCES

Brinkley, M. A brief survey of methods for preparing protein conjugateswith dyes, haptens, and cross-linking reagents. Bioconjug. Chem. 3:2-13(1992).

Carlsson, J., Drevin, H., and Axen, R. Protein thiolation and reversibleprotein-protein conjugation. N-Succinimidyl3-(2-pyridyldithio)propionate, a new heterobifunctional reagent.Biochem. J. 173(3):723-737 (1978).

Duncan, R. J., Weston, P. D., Wrigglesworth, R. A new reagent which maybe used to introduce sulfhydryl groups into proteins, and its use in thepreparation of conjugates for immunoassay. Anal. Biochem. 132(1):68-73(1983).

Eyzaguirro, J. CHEMICAL MODIFICATION OF ENZYMES: ACTIVE SITE STUDIES.John Wiley & Sons, New York (1987).

Hope, M. J., Bally, M. B., Webb, G. and Cullis, P. R. Production oflarge unilamellar vesicles by a rapid extrusion procedure.Characterization of size distribution, trapped volume and ability tomaintain a membrane potential. Biochim. Biophys. Acta. 812:55-65 (1985).

Mattson, G., Conklin, E., Desai, S., Nielander, G., Savage, M. D. andMorgensen, S. A practical approach to crosslinking. Mol. Biol. Rep.17(3):167-183 (1993).

Means, G. E. and Feeney, R. E. Chemical modifications of proteins:history and applications. Bioconjug. Chem. 1:2-12 (1990).

Wong, S. H. CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING. CRCPress, Boca Raton, Fla. (1991).

Wong, S. S. and Wong, L. J. Chemical crosslinking and the stabilizationof proteins and enzymes. Enzyme Microb. Technol. 14:866-874 (1992).

BACKGROUND OF THE INVENTION

Scientists and clinicians face a number of challenges in development oftherapeutically active agents into forms suitable for delivery to apatient. For example, pharmaceutically useful biomolecules, includingantibodies and antibody fragments, can now be prepared on a usefulscale, due to advances in biotechnology, but the clinical usefulness ofpotentially therapeutic biomolecules is often hampered by their rapidproteolytic degradation, their instability upon manufacture, storage oradministration, and/or their immunogenicity. Therapeutically potentmolecules can be hampered by low aqueous solubility; paclitaxel (Taxol™)is one significant example.

Conjugation of active agents to water-soluble polymers has been found toreduce immunogenicity and antigenicity, as well as increasing half-lifein circulation, as a result of decreased clearance and/or decreasedenzymatic degradation in systemic circulation. The frequency ofadministration can thus be reduced, which is particularly beneficial inthe large number of cases in which the agent is administered byinjection. As a further benefit, active agents that are only marginallysoluble in water often demonstrate a significant increase in watersolubility when conjugated to a water soluble polymer.

Polyethylene glycol, due to its documented safety and its approval bythe FDA for both topical and internal use, has been conjugated to avariety of active agents. Such a conjugated active agent isconventionally referred to as “PEGylated.” Commercially successfulPEGylated active agents include PEGASYS® PEGylated interferon α-2a(Hoffmann-La Roche, Nutley, N.J.), PEG-INTRON® PEGylated interferon α-2b(Schering Corp., Kennilworth, N.J.), NEULASTA™ PEG-filgrastim (AmgenInc., Thousand Oaks, Calif.) and SOMAVERT® pegvisomant, a PEGylatedhuman growth hormone receptor antagonist (Pfizer, New York, N.Y.).Non-peptidic small molecules such as fluorouracil (Ouchi et al., DrugDes. Discov. 9(1):93-105, 1992) have also been prepared in PEGylatedform.

In view of these promising features, there has been an increasing needfor high purity functionalized derivatives of PEG and other watersoluble polymers having medium to high molecular weight. However, thesynthesis of such compounds is often complicated by the difficulty inremoving polymeric impurities that accumulate during multi-steppreparations. For example, end-capped (i.e., nominally monofunctional)polyethylene glycol starting material often contains significant amountsof PEG diol impurity, ranging from 0.5% to over 30% by weight. The diolimpurity, and especially its reaction products when carried through aseries of synthetic transformations, can be extremely difficult toanalyze and remove. Higher molecular weight polymeric side-products, inparticular, are generally quite difficult to remove and requiretime-consuming and expensive chromatographic techniques. Accordingly,there remains a need in the art for improved methods of preparingfunctionalized derivatives of water soluble polymers such as PEG.

SUMMARY OF THE INVENTION

The invention provides segmented, functionalized water soluble polymerderivatives and methods of their preparation. In one aspect, theinvention provides a water soluble polymer, where the polymer comprises:

(i) a first water soluble polymer segment having at least 4 and at mostabout 2000 monomeric units (designated “POLY_(A)”);

(ii) a second water soluble segment comprising 1 to about 120 monomericunits (designated “POLY_(B)”), which has a lower molecular weight thanthat of POLY_(A) and is covalently attached to POLY_(A) through alinkage X, as described further below; and

(iii) a functional group, Y, attached to POLY_(A) or POLY_(B). Afunctional group may be located on either one or both of POLY_(B) andPOLY_(A). Typically, a functional group is located on POLY_(B).

Typically, POLY_(B) has fewer monomeric units, as well as a lowermolecular weight, than POLY_(A). The molecular weight of the POLY_(A)segment is preferably at least two times, and more preferably at leastfour times, that of the POLY_(B) segment (excluding functional groups).The molecular weight of the POLY_(A) segment may also be at least ten orat least twenty times that of the POLY_(B) segment (excluding functionalgroups).

Each of the water soluble segments designated POLY_(A) and POLY_(B)independently comprises one monomer, or up to three different monomers,selected from the group consisting of alkylene glycol, olefinic alcohol,vinyl pyrrolidone, hydroxyalkyl methacrylamide, hydroxyalkylmethacrylate, saccharide, α-hydroxy acid, phosphazene, oxazoline, andN-acryloylmorpholine. That is, each segment is, independently, ahomopolymer of one monomer selected from this group, a binary copolymercomprising two different monomers selected from this group, or aterpolymer comprising three different monomers selected from this group;homopolymers and binary copolymers are generally preferred. Thedifferent monomers may be of the same monomer type; for example, twoalkylene glycols, such as ethylene glycol and propylene glycol.Preferably, POLY_(A) and POLY_(B) have the same monomeric composition,with the possible exception of attached functional groups and/or cappinggroups.

In selected embodiments, each of POLY_(A) and POLY_(B) is independentlya poly(alkylene glycol). For example, each of POLY_(A) and POLY_(B) maybe selected from poly(ethylene glycol) and an ethylene glycol/propyleneglycol copolymer. In preferred embodiments, each of POLY_(A) andPOLY_(B) is a poly(ethylene glycol).

POLY_(A) may be a “high” molecular weight segment, i.e. having more than200 monomeric repeating units, or a “medium” molecular weight segment,i.e. having 4 to about 200 monomeric repeating units. In selectedembodiments, a “high molecular weight” POLY_(A) segment has at least200, 250, 500, 1000, 1500, or 2000 or more monomeric units. These rangescorrespond, for example, to poly(ethylene glycol) segments havingmolecular weights of at least about 8800, 11,000, 22,000, 44,000,66,000, or 88,000 or more Daltons, respectively.

In other selected embodiments, POLY_(A) is a “medium” molecular weightsegment having at least 10, 20, 25, 50, 100, 125, 150, or 175 monomericunits, up to about 200 monomeric units. These ranges correspond, forexample, to poly(ethylene glycol) segments having molecular weights ofat least about 440, 880, 1100, 2200, 4400, 5500, 6600, and 7700 Daltons,respectively, up to about 8800 Daltons.

In selected embodiments of segmented PEG-based polymers, where each ofPOLY_(A) and POLY_(B) is a poly(ethylene glycol) segment, POLY_(A) has amedium molecular weight range selected from about 200-5000, about500-2000, and about 1000-1500. In other embodiments of PEG-basedsegmented polymers, POLY_(A) has a high molecular weight range selectedfrom about 8800 to about 20000, about 15000 to about 50000, and about20000 to about 90000.

Preferably, the segment POLY_(B) has at least two monomeric units; morepreferably, POLY_(B) has at least three monomeric units. In otherpreferred embodiments, POLY_(B) has at most about 100, and morepreferably at most 50, monomeric units. In selected embodiments,POLY_(B) has 2, 3, 4, 5, 10, 15, 25, 35, 45, 75 or 100 monomeric units.These ranges correspond, for example, to poly(ethylene glycol) segmentshaving molecular weights of about 88, 132, 176, 220, 440, 660, 1100,1540, 1980, 3300, and 4400 Daltons, respectively.

In selected embodiments of segmented PEG-based polymers, where each ofPOLY_(A) and POLY_(B) is a poly(ethylene glycol) segment, POLY_(B)preferably has a molecular weight range selected from 44 to about 4400,44 to about 1500, 220 to about 2000, and about 2000 to about 3300.

The linkage X between the polymer segments is distinct from themonomeric units of the polymer segments; that is, there is a cleardelineation between the structure of the linkage and the structure ofthe adjacent portions of the polymer segments. In selected embodiments,X comprises an amide, a carbamate, a carbonate, a urea, an ester, anamine, a thioether, or a disulfide; in further selected embodiments, Xcomprises an amide, a carbonate, or a carbamate. The linkage istypically formed by reaction between functional groups on precursorsegments, as described further below. The linkage may also comprisespacer groups, as described further below.

The functional group Y preferably comprises a moiety selected fromhydroxyl, amine, hydrazine, hydrazide, thiol (nucleophilic groups),carboxylic acid, carboxylic ester, including imide ester, orthoester,carbonate, isocyanate, isothiocyanate, aldehyde, ketone, thione,alkenyl, acrylate, methacrylate, acrylamide, sulfone, maleimide,disulfide, iodo, epoxy, sulfonate, thiosulfonate, silane, alkoxysilane,halosilane, and phosphoramidate. More specific examples of these groupsinclude succinimidyl ester or carbonate, imidazolyl ester or carbonate,benzotriazole ester or carbonate, p-nitrophenyl carbonate, vinylsulfone, chloroethylsulfone, vinyipyridine, pyridyl disulfide,iodoacetamide, glyoxal, dione, mesylate, tosylate, and tresylate. Alsoincluded are other activated carboxylic acid derivatives, as well ashydrates or protected derivatives of any of the above moieties (e.g.aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal). Preferred electrophilic groups includesuccinimidyl carbonate, succinimidyl ester, maleimide, benzotriazolecarbonate, glycidyl ether, imidazoyl ester, p-nitrophenyl carbonate,acrylate, tresylate, aldehyde, and orthopyridyl disulfide.

When Y is a nucleophile, preferred nucleophiles include amine,hydrazine, hydrazide (i.e. —C(═O)NHNH₂) and thiol, particularly amine.

Particular structural embodiments of Y, where the functional group maybe attached to an alkyl spacer group, include structures of the form—(CH₂)_(r)CO₂Q, where Q is selected from N-succinimide,N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole. Also included are groups of the form—(CH₂)_(r)CH(OR)₂, —(CH₂)_(r)CHO, —(CH₂)_(r)NH₂, and —(CH₂)_(r)M, whereM is N-maleimide. In these structures, r is 0-5, preferably 1-5, andmore preferably 2-3.

In selected embodiments of the segmented polymer, the functional group Ycomprises an amine or protected amine, and is preferably present onPOLY_(B), and the linkage X comprises a carbamate, an amide, a urea, ora carbonate. In further preferred embodiments of this group, each ofPOLY_(A) and POLY_(B) is independently a poly(ethylene glycol). Invarious embodiments, POLY_(A) has a molecular weight of about 200 to40,000, 200 to 10,000, or 200-1000; in one embodiment, POLY_(A) has amolecular weight of about 1000. Preferably, POLY_(A) is end-capped witha C₁-C₂₀ alkoxy group, more preferably with a C₁-C₁₂ alkoxy group, e.g.a methoxy group.

In one embodiment, the segmented polymer is linear (i.e., each ofPOLY_(A) and POLY_(B) is linear) and comprises a functional group ateach terminus. The functional groups may be the same (homobifunctional)or different (heterobifunctional). Alternatively, the segmented polymeris linear and comprises a functional group at one terminus, e.g. onPOLY_(B), and a capping group, such as alkoxy, on the other terminus.Such polymers are referred to herein as “monofunctional”.

In a further embodiment, the segmented polymer comprises two POLY_(B)segments and has the structure Y-POLY B-X-POLY A-X-POLY B-Y, where eachX and each Y is independently selected, and X and Y are as definedabove. In a preferred embodiment, each X in the structure above is thesame and each Y is the same.

In preferred embodiments, the invention provides a conjugate formed byattachment of one or more pharmacologically active agents to theabove-described polymer, typically via the functional group(s) Y. Theagent(s) may include, for example, proteins, peptides, carbohydrates,oligonucleotides, DNA, RNA, lipids, or small molecule compounds, asdiscussed further below.

In further embodiments, each of POLY_(A) or POLY_(B) may behydrolytically or enzymatically degradable, in that each segmentindependently comprises at least one hydrolytically or enzymaticallydegradable linkage separating monomeric units of the polymer segment.Alternatively, or in addition, the linkage(s) joining POLY_(A) andPOLY_(B) segments may be hydrolytically or enzymatically degradable.

Each of POLY_(A) and POLY_(B) may be independently selected from thegroup consisting of linear, branched, forked, multi-amied, anddendrimeric polymer segments, as defined further below. The segmentedpolymer comprising POLY_(A) and POLY_(B) may also have an architectureselected from linear, branched, forked, multi-armed, and dendrimeric.

For example, the polymer may be a linear polymer, having the generalstructure

POLY_(A)-X-POLY_(B)-Y   (Ia)

or

POLY_(B)-X-POLY_(A)-Y   (Ib)

where structure Ia is generally preferred. In further selectedembodiments, a linear segmented PEG polymer is provided, having thestructure:

R—(CH₂CH₂O)_(n)—CH₂CH₂—X—(CH₂CH₂O)_(m)—CH₂CH₂—Y   (Ic)

where:

R is a functional group or capping group;

n is at least 4 and at most about 2000,

m is between 1 and about 120, where m<n,

X is a linking group, as described herein, and

Y is a functional group, as described herein, which may be the same ordifferent from R when R is a functional group. In selected embodiments,n is at most about 1000, at most about 500, or at most about 200.

When R is a capping group, it is preferably selected from C₁-C₂₀ alkoxyor aryloxy, more preferably C₁-C₁₀ alkoxy or aryloxy, and aphospholipid. In further selected embodiments, R is C₁-C₅ alkoxy orbenzyloxy. A preferred phospholipid is a dialkylphosphatidylethanolamine, such as distearoyl phosphatidylethanolamine(DSPE). The terminal amine of the head group may be used to link thelipid to the PEG chain via, for example, a carbamate linkage.

Alternatively, the polymer may be a branched polymer, having a generalstructure selected from

(POLY_(A)-X-POLY_(B))_(p)-L-Y   (IIa)

(POLY_(A)-X)_(p)-L-POLY_(B)-Y   (IIb)

and

(POLY_(A))_(p)-L-X-POLY_(B)-Y   (IIc)

where POLY_(A), POLY_(B), X, and Y are as defined above; L represents abranched spacer group; and p is ≧2, preferably 2 to about 100, morepreferably 2 to about 10. In one embodiment, p is 2.

Preferably, the branch point in the branched spacer group L comprises acarbon atom (—CH<), though it may also comprise a nitrogen atom (—N<).The branch point is linked to adjacent moieties, such as X, Y or apolymer segment, directly or by chains of atoms of defined length. Eachchain of atoms may comprise, for example, alkyl, ether, ester, or amidelinkages, or combinations thereof.

In selected embodiments of structures IIa-c above, the POLY_(A) segmentis a PEG segment (PEG_(A)) comprising at least 4 and at most about 2000—CH₂CH₂O— monomeric units; X is selected from an amide, a carbamate, acarbonate, an ester, a urea, an amine, a thioether, and a disulfide; andthe POLY_(B) segment is a PEG segment (PEG_(B)) comprising at least 1,preferably at least 2, and at most about 120 —CH₂CH₂O— monomeric units,with the proviso that PEG_(B) has a lower molecular weight than that ofPEG_(A). Preferred ranges of p include 1-100, 2-20, and 2-10.

Also provided are “forked” polymers, in which a single segmented polymer(which can itself be, for example, linear or branched) is linked to twofunctional groups, or, in a conjugate, to two pharmacologically activeagents, as in the following general structures:

POLY_(A)-L-(X-POLY_(B)-Y)_(q)   (IIIa)

POLY_(A)-X-L-(POLY_(B)-Y)_(q)   (IIIb)

POLY_(A)-X-POLY_(B)-L-(Y)_(q)   (IIIc)

where POLY_(A), POLY_(B), X, and Y are as defined above; L represents abranched spacer group; and q is ≧2, preferably 2 to about 100, morepreferably 2 to about 10. In one embodiment, q is 2. Preferably, thebranch point in the spacer group comprises a carbon atom (>CH—), thoughit may also comprise a nitrogen atom (>N—). A “multiarmed” polymer ofthe invention comprises three or more water-soluble polymer chainsattached to a central core structure, and can be represented by thegeneral structure:

M-{POLY_(A)-X-(POLY_(B)-Y)_(q)}_(x)   (IV)

where M is a core structure, having several linkage points forattachment of POLY_(A). The value of x is typically 3 to about 100, moretypically 3 to about 20. The core structure M is preferably selectedfrom a polyol, a polyamine, and an amino acid whose side chain bears afunctional group, and more preferably selected from a polyol and apolyamine.

In selected embodiments of structure (IV), q is 1 or 2; that is, each“arm” represents a linear polymer (q=1) or a “forked” polymer (q=2). Infurther embodiments, x is 3 to 20, or 3 to 10. Generally, q is 1, suchthat each POLY_(A) is linked to a single POLY_(B). However, q can begreater than 1, and POLY_(B) can include more than one Y or apolyfunctional Y, as noted above. Where q is >1 or POLY_(B) includesmore than one Y, branched spacer groups, as represented by L above, areincorporated as appropriate. Generally, POLY_(B) includes a single groupY.

The multiarmed polymer may also include some POLY_(A) segments which arenot further substituted. Accordingly, structure (IV) can represent abranched or dendritic polymer segment in which each of a plurality ofPOLY_(A) polymer arms (where the plurality is equal to or less than q)is covalently attached to a POLY_(B)-Y segment via a covalent linkage,X.

In selected embodiments, the multiarmed polymer is a PEG polymer, e.g.:

M-{PEG_(A)-X-(PEG_(B)-Y)_(q)}_(x)   (IV′)

where PEG_(A) represents poly(ethylene glycol) comprising at least 4 andat most about 2000 —CH₂CH₂O— monomeric units, and PEG_(B) representspoly(ethylene glycol) comprising at least 1 and at most about 120—CH₂CH₂O— monomeric units, with the proviso that PEG_(B) has a lowermolecular weight than that of PEG_(A). In selected embodiments, Xcomprises a carbamate, an amide, a carbonate, an ester, a urea, anamine, a thioether, or a disulfide. Preferably, X comprises a carbamate,an amide, a urea, or a carbonate.

The invention also provides gels, also referred to as hydrogels, whichmay be crosslinked or uncrosslinked, comprising a water soluble,segmented polymer as described above.

In another aspect, the invention provides a method of forming awater-soluble polymer comprising two or more segments, as describedabove. The method comprises reacting a first water soluble polymersegment, having at least 4 and at most about 2000 monomeric units, andhaving at least one first functional group, Z, with a second watersoluble segment, having from 1 to about 120 monomeric units and havingat least one second functional group, Y′, thereby covalently bonding thefirst and second segments by reaction of Z with Y′. Either the first orsecond segment, preferably the second segment, further comprises anadditional functional group, Y, that is not readily reactive with eitherZ or Y′.

The second segment has a lower molecular weight than the first segment,as described for POLY_(B) and POLY_(A) above. As also described forPOLY_(A) and POLY_(B) above, each of the first and second segmentsindependently comprises up to three different monomers which areselected from the group consisting of alkylene glycol, olefinic alcohol,vinylpyrrolidone, hydroxyalkylmethacrylamide, hydroxyalkylmethacrylate,saccharides, α-hydroxy acid, vinyl alcohol, polyphosphazene,polyoxazoline, and N-acryloylmorpholine.

Selected embodiments of size and molecular weight for the first andsecond segments are also as described for POLY_(A) and POLY_(B) above.In preferred embodiments, each of the first and second segments is apoly(alkylene glycol), such as PEG. In one embodiment, the first polymersegment is a linear methoxypoly(ethylene glycol).

Preferably, each of the reacting groups Z and Y′ independently comprisesa group selected from hydroxy, thiol, amine, hydrazide, hydrazide,N-succinimidyl carbonate, succinimidyl ester, benzotriazole carbonate,glycidyl ether, imidazolyl ester, aldehyde, maleimide, ortho-pyridyldisulfide, acrylate, and vinyl sulfone. The groups are generallyselected such that one is nucleophilic (e.g. hydroxy, thiol, amine,hydrazide) and the other is electrophilic.

The remaining functional group Y preferably comprises a moiety selectedfrom hydroxyl, amine, hydrazine, hydrazide, thiol (nucleophilic groups),carboxylic acid, carboxylic ester, imide ester, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, halosilane, and phosphoramidate. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate. Also included are various activated carboxylic acidderivatives, as well as hydrates or protected derivatives of any of theabove moieties (e.g. aldehyde hydrate, hemiacetal, acetal, ketonehydrate, hemiketal, ketal, thioketal, thioacetal). Preferredelectrophilic groups include succinimidyl carbonate, succinimidyl ester,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl ester,p-nitrophenyl carbonate, acrylate, aldehyde, and orthopyridyl disulfide.When Y is a nucleophile, preferred nucleophiles include amine,hydrazine, hydrazide, and thiol, particularly amine. The group Y may bepresent in protected form, particularly as required to prevent reactionwith Z or Y′.

The method may further comprises the step of conjugating apharmacologically active agent to the segmented polymer as describedabove, typically via the functional group Y.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to a “polymer” includes a single polymer aswell as two or more of the same or different polymers, reference to a“conjugate” refers to a single conjugate as well as two or more of thesame or different conjugates, reference to an “excipient” includes asingle excipient as well as two or more of the same or differentexcipients, and the like.

“Water soluble” indicates that a polymer or polymer segment is at least35% (by weight) soluble, and preferably greater than 95% soluble, inwater at room temperature. Typically, an unfiltered aqueous preparationof a “water soluble” polymer or segment transmits at least 75%, morepreferably at least 95%, of the amount of light transmitted by the samesolution after filtering. On a weight basis, a “water soluble” polymeror segment is preferably at least 35% (by weight) soluble in water, morepreferably at least 50% (by weight) soluble in water, still morepreferably at least 70% (by weight) soluble in water, and still morepreferably at least 85% (by weight) soluble in water. It is mostpreferred, however, that the water-soluble polymer or segment is atleast 95% (by weight) soluble in water or completely soluble in water.

As used herein, a “segment” can consist of a single monomeric unit, e.g.—(CH₂CH₂O)—. Typically, however, a “polymer segment” contains multiplemonomeric units. The “polymer segment” may be oligomeric; that is,including two to about ten monomeric units.

A “segmented polymer” refers to a polymer comprising at least twopolymer segments joined by one or more linkages as defined herein, thelinkage(s) being distinct from the monomeric units of the polymersegments.

A “monomeric unit” refers to one of the basic structural units of apolymer segment. In the case of homopolymeric segments, this can bedefined as a structural repeating unit of the polymer segment. In thecase of copolymers, a monomeric unit is more usefully defined as theresidue of a monomer which was polymerized to form the polymer segment,since the structural repeating unit can include more than one monomericunit. “PEG” or “polyethylene glycol,” as used herein, is meant toencompass any water-soluble poly(ethylene oxide). A “PEG polymer” canrefer to a polymer having monomeric subunits of which greater than 50%are ethylene oxide (—CH₂CH₂O—) subunits; preferably, it refers to apolymer having monomeric subunits of which greater than 75%, greaterthan 90%, or greater than 95%, are ethylene oxide subunits. In oneembodiment, substantially all, or all, monomeric subunits are ethyleneoxide subunits, though the polymer may contain distinct end cappingmoieties, or functional groups, e.g. for conjugation. Typically, PEGsfor use in the present invention will comprise one of the two followingstructures: “—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” dependingupon whether or not the terminal oxygen(s) has been displaced, e.g.,during a synthetic transformation. The variable (n) is 3 to 3000, andthe terminal groups and architecture of the overall PEG may vary. WhenPEG further comprises a linker moiety (to be described in greater detailbelow), the atoms comprising the linker (X′), when covalently attachedto a PEG segment, do not result in formation of (i) an oxygen-oxygenbond (O—O, a peroxide linkage), or (ii) a nitrogen-oxygen bond (N—O,O—N).

A PEG polymer having non-(ethylene oxide) monomeric units is morepreferably referred to as a PEG copolymer. Random or block copolymers ofethylene oxide and propylene oxide are a common example.

End-capped PEGs are commonly employed. The end capping group isgenerally a carbon-containing group attached to a terminal oxygen,typically comprised of 1-20 carbons and preferably alkyl or aralkyl(e.g., methyl, ethyl or benzyl), although saturated and unsaturatedforms of these groups, as well as aryl, heteroaryl, cycloalkyl,heterocycle, and substituted forms of any of the foregoing are alsoenvisioned. The end capping group may alternatively be a phospholipid. Apreferred phospholipid is a dialkyl phosphatidyl ethanolamine, such asdistearoyl phosphatidylethanolamine (DSPE), where the terminal amine ofthe phosphate head group can be used to link the lipid to the PEG chainvia, for example, a carbamate linkage. Other suitable phospholipidsinclude, for example, phosphatidyl cholines and phosphatidyl inositols.The end-capping group can also comprise a detectable label. Such labelsinclude, without limitation, fluorescers, chemiluminescers, moietiesused in enzyme labeling, colorimetric labels (e.g., dyes), metal ions,and radioactive moieties. The other (uncapped) terminus is a typicallyhydroxyl or another functional group, e.g. amine, that can be subjectedto further chemical modification.

The architecture of a PEG molecule may vary. Specific PEG forms for usein the invention include PEGs having a variety of structures orgeometries (e.g., branched, linear, forked, multiarmed, dendrimeric, andthe like), to be described in greater detail below.

“Branched”, in reference to the geometry or overall structure of apolymer, refers to a polymer having 2 or more polymer “arms”. A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, which, for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer or linking group splits or branches from alinear structure into one or more additional polymer arms.

A “dendrimer” is a globular, size monodisperse polymer in which allbonds emerge radially from a central focal point or core with a regularbranching pattern and with repeat units that each contribute a branchpoint. Dendrimers exhibit certain dendritic state properties such ascore encapsulation, distinguishing them from other types of polymers.

“Substantially” or “essentially” means nearly totally or nearlycompletely, for instance, 95% or greater of some given quantity.

“Non-naturally occurring”, with respect to a polymer of the invention,refers to a polymer that is not found in nature in its entirety,although it may contain one or more subunits or segments of subunitsthat are naturally occurring.

“Molecular mass” or “molecular weight” refers to the nominal averagemolecular mass of a polymer, typically determined by size exclusionchromatography, light scattering techniques, or intrinsic velocitydetermination in 1,2,4-trichlorobenzene. The polymer segments of theinvention are typically polydisperse, possessing low polydispersityvalues of less than about 1.20.

The “linkage” or “linking group” between polymer segments, representedby X, refers to a linkage that is distinct from the monomeric units ofthe polymer segments. That is, there is a clear delineation between thestructure of the linkage and the structure of the adjacent portions ofthe polymer segments. Typically, the linkage is a group formed byreaction between reactive functional groups on the precursor polymersegments; e.g. a nucleophilic functional group on the first polymersegment and an electrophilic group on the second polymer segment.

A “functional group” is a group that may be used, under normalconditions of organic synthesis, to form a covalent linkage between thestructure to which it is attached and another structure, which typicallybears a further functional group. The functional group generallyincludes multiple bond(s) and/or heteroatom(s). Preferred functionalgroups for use in the polymers of the invention are described below.

The term “reactive” refers to a functional group that reacts readily orat a practical rate under conventional conditions of organic synthesis.This is in contrast to those groups that either do not react or requirestrong catalysts or impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

“Not readily reactive”, with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions effective to produce a desired reactionin the reaction mixture.

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative which reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters include, forexample, imidazolyl esters, and benzotriazole esters, and imide esters,such as N-hydroxysuccinimidyl (NHS) esters. An activated derivative maybe formed in situ by reaction of a carboxylic acid with one of variousreagents, e.g. benzotriazol-1-yloxy tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), preferably used in combination with1-hydroxy benzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT);O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU); or bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BOP-Cl).

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

The term “spacer” or “spacer moiety” refers to an atom or, moretypically, a collection of atoms, used to link interconnecting moieties,such as the terminus of a polymer segment and a functional group orlinking group. The spacer moieties of the invention may behydrolytically stable, including bonds such as alkyl, ether, or amide.Particularly preferred are alkyl or alkyl ether spacer groups.Alternatively, they may include a physiologically hydrolyzable orenzymatically degradable linkage, such as an ester or disulfide.

“Alkyl” refers to a saturated hydrocarbon chain, typically ranging fromabout 1 to 20 carbon atoms in length, more typically about 1 to 12carbon atoms in length. Such hydrocarbon chains may be branched or, moretypically, linear. Exemplary alkyl groups include ethyl, propyl, butyl,pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. Asused herein, “alkyl” includes cycloalkyl having three or more carbonatoms.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, preferably 1 to 4 carbon atoms, and may be linear or branched, asexemplified by methyl, ethyl, isopropyl, isobutyl, n-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonradical, including bridged, fused, or spiro cyclic compounds, preferablyhaving 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Non-interfering substituents” are groups that are typicallynon-reactive with other functional groups contained within the samemolecule, under normal conditions of organic synthesis.

The term “substituted”, as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to, C₃-C₈cycloalkyl; halo, e.g., fluoro, chloro, bromo, or iodo; cyano; nitro;alkoxy; hydroxy; amino; alkylamino; carboxylic acid or ester; sulfonicacid or ester; phenyl; substituted phenyl; and the like. “Substitutedaryl” refers to aryl having one or more non-interfering substituents inany orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —OR group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), more preferably C₁-C₇.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbonhaving 2 to about 15 carbon atoms and containing at least one doublebond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon having 2 to about 15 carbon atoms and containing at leastone triple bond, such as ethynyl, n-propynyl, isopropynyl, n-butynyl,isobutynyl, octynyl, or decynyl.

The term “aryl” refers to a monovalent aromatic hydrocarbon having asingle ring (i.e., phenyl) or fused rings (i.e., naphthalene). Unlessotherwise defined, such aryl groups typically contain from 4 to 10carbon ring atoms. Multiple aryl rings may be fused, as in naphthyl, orunfused (linked), as in biphenyl. Aryl rings may also be fused or linkedwith one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings.Representative aryl groups include, by way of example, phenyl,naphthalene-1-yl, naphthalene-2-yl, and the like.

“Aralkyl” refers to an alkyl, preferably lower (C₁-C₄, more preferablyC₁-C₂) alkyl, substituent which is further substituted with an arylgroup; examples are benzyl and phenethyl.

As used herein, “aryl” includes heteroaryl. The term “heteroaryl” refersto a monovalent aromatic group containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Heteroaryl rings may also be fused with one or more cyclichydrocarbon, heterocyclic, aryl, or heteroaryl rings. Unless otherwisedefined, such heteroaryl groups typically contain from 5 to 10 totalring atoms. Representative heteroaryl groups include, by way of example,monovalent species of pyrrole, imidazole, thiazole, oxazole, furan,thiophene, triazole, pyrazole, isoxazole, isothiazole, pyridine,pyrazine, pyridazine, pyrimidine, triazine, indole, benzofuran,benzothiophene, benzimidazole, benzthiazole, quinoline, isoquinoline,quinazoline, quinoxaline and the like.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” refers to heteroaryl having one or morenon-interfering groups as substituents.

A “heterocycle” refers to a non-aromatic ring, preferably a 5- to7-membered ring, whose ring atoms are selected from the group consistingof carbon, nitrogen, oxygen and sulfur. Preferably, the ring atomsinclude 3 to 6 carbon atoms. Such heterocycles include, for example,pyrrolidine, piperidine, piperazine, and morpholine.

A “substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents,

An “electrophile” refers to an atom or collection of atoms, which may beionic, having an electrophilic center, i.e., a center that is electronseeking, capable of reacting with a nucleophile.

A “nucleophile” refers to an atom or collection of atoms, which may beionic, having a nucleophilic center, i.e., a center that seeks anelectrophilic center, capable of reacting with an electrophile.

A “physiologically hydrolyzable” bond is a bond that reacts with water(i.e., is hydrolyzed) under physiological conditions. Such a bond istherefore degradable in water or aqueous media, e.g. blood, underphysiological conditions. Such bonds generally include, for example,carboxylate esters, phosphate esters, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, orthoesters, and oligonucleotide bonds.

An “enzymatically degradable linkage” is a linkage that is subject todegradation by one or more enzymes. Such bonds generally include, forexample, carboxylate esters, phosphate esters (e.g. oligonucleotidelinkages), and peptide bonds.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water; i.e.it does not undergo hydrolysis under physiological conditions to anyappreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, for example, carbon-carbon bonds(e.g., in aliphatic chains), ethers, amines, and, to a lesser extent,amides. Generally, a hydrolytically stable linkage is one that exhibitsa rate of hydrolysis of less than about 1-2% per day under physiologicalconditions. Hydrolysis rates of representative chemical bonds can befound in most standard chemistry textbooks.

“Multifunctional”, in the context of a polymer of the invention, refersto a polymer having 2 or more, preferably 3 or more, functional groups,which may be the same or different. Multifunctional polymers of theinvention will typically contain from about 3-100 functional groups, orfrom 3-50 functional groups, or from 3-25 functional groups, or from3-15 functional groups, or from 3 to 10 functional groups, or willcontain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the polymerbackbone. A “bifunctional” polymer is a polymer having two functionalgroups, either the same (i.e., homobifunctional) or different (i.e.,heterobifunctional).

Reference to a compound which is basic or acidic is typically intendedto encompass neutral, charged, and any corresponding salt forms thereof.It will be apparent to those skilled in the art when a particular formis most suitable for a desired purpose, such as a desired reaction orfor purposes or solubility.

A “hydrogel” is a material that absorbs a solvent (e.g. water),undergoes rapid swelling without discernible dissolution, and maintainsthree-dimensional networks capable of reversible deformation. Hydrogelsmay be uncrosslinked or crosslinked. Covalently (chemically) crosslinkednetworks of hydrophilic polymers, such as PEG, can form hydrogels (oraquagels) in the hydrated state. Uncrosslinked hydrogels are typicallyblock copolymers having hydrophilic and hydrophobic regions. Theseuncrosslinked materials can form hydrogels when placed in an aqueousenvironment, due to physical crosslinking forces resulting from ionicattractions, hydrogen bonding, Van der Waals forces, etc. They are ableto absorb water but do not dissolve due to the presence of hydrophobicand hydrophilic regions.

A “polymer conjugate” as used herein refers to a water soluble segmentedpolymer covalently attached to a further moiety, such as apharmacologically active molecule or surface.

A “pharmacologically active” agent includes any drug, compound,composition of matter or mixture desired to be delivered to a subject,e.g. therapeutic agents, diagnostic agents, or drug delivery agents,including targeting agents, which provides or is expected to providesome pharmacologic, often beneficial, effect that can be demonstrated invivo or in vitro. Such agents may include biomolecules, e.g. peptides,proteins, carbohydrates, nucleic acids, nucleosides, oligonucleotides,and lipids, or analogs thereof, as well as dyes, liposomes,microparticles, and therapeutic “small molecule” compounds. Examples oflipids include phospholipids, glycolipids, such as cerebrosides andgangliosides, sphingolipids, fatty diacylglycerides, triglycerides,glycosylglycerides, and steroids, including sterols.

A “small molecule” compound may be defined broadly as an organic,inorganic, or organometallic compound which is not a biomolecule asdescribed above. Typically, such compounds have molecular weights ofless than about 1000.

Classes of therapeutic agents that are suitable for use with theinvention include, but are not limited to, antibiotics, fungicides,anti-viral agents, anti-inflammatory agents, anti-tumor agents,cardiovascular agents, anti-anxiety agents, hormones, growth factors,steroidal agents, and the like.

A “pharmaceutically acceptable excipient” or “pharmaceuticallyacceptable carrier” refers to an excipient that can be included in thecompositions of the invention and that causes no significant adversetoxicological effects to the subject or patient to which the compositionis administered. “Pharmacologically effective amount,” “physiologicallyeffective amount,” and “therapeutically effective amount” are usedinterchangeably herein to mean the amount of a PEG-active agentconjugate present in a pharmaceutical preparation that is needed toprovide a desired level of active agent and/or conjugate in thebloodstream or in the target tissue. The precise amount will depend uponnumerous factors, e.g., the particular active agent, the components andphysical characteristics of pharmaceutical preparation, intended patientpopulation, patient considerations, and the like, and can readily bedetermined by one skilled in the art, based upon the informationprovided herein and available in the relevant literature.

The term “patient” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of apolymer-agent conjugate as described herein, and includes both humansand animals.

II. Segmented Polymers of the Invention

The invention includes medium to high molecular weight, functionalized,water soluble segmented polymers and methods for preparing them. Thepolymers of the invention include a “medium to high” molecular weightsegment, which may be designated POLY_(A), which imparts desiredphysical and pharmacological properties to a conjugate of the polymerwith a phannacological agent. This segment is covalently linked to a“low molecular weight” segment, which may be designated POLY_(B),typically having a composition similar to POLY_(A). In preparing theconjugate, as described further below, synthetic transformations such asattachment of functional groups, linker moieties, and/or spacer groupsand purification steps are advantageously carried out on the POLY_(B)segment, which is then linked, at a later stage in the preparation, toPOLY_(A).

Large polymeric impurities are more difficult to separate from thedesired product than are smaller ones, and the products of thesereactions involving these polymers typically include unreacted reagents,bifunctional components that can result in cross linking, partiallyreacted components, and other polymeric impurities. The syntheticstrategy described herein reduces the number of synthetictransformations carried out on POLY_(A) and thus reduces the need forlaborious purification of higher molecular weight intermediates.

A. Size and Molecular Weight Ranges

The molecular weight of the POLY_(B) segment is generally low, e.g.preferably below 5000, and more preferably below 2000, for purposes ofsynthesis and purification, as discussed above. The molecular weight ofthe POLY_(A) segment, and the overall polymer, are selected inaccordance with the intended use of the polymer conjugate.

For example, it is well established that higher molecular weight polymerconjugates generally provide longer circulation times, as well assignificant tumor accumulation (e.g., Y. Murakami et al., Drug. Del.4:23-32, 1997), when used for drug delivery applications. However, insome cases, such as administration of thrombolytic drugs, or manydiagnostic applications, long circulation times may be less desirable.In addition, the use of a lower molecular weight polymer (such as the“medium” molecular weight ranges for PEG described herein) can providegreater drug loading for a smaller mass of conjugate administered.Larger polymers may also be more likely to block reactive sites ofattached drugs (although this can frequently be addressed via the use ofcleavable conjugates). In view of these factors, the preferred molecularweight of POLY_(A) can vary widely.

Preferably, the number of monomeric units in POLY_(A) is between 4 andabout 2000, and the number of monomeric units in POLY_(B) is between 1and about 120, as long as POLY_(B) has a lower molecular weight thanthat of POLY_(A). Typically, POLY_(B) will have fewer monomeric units,as well as a lower molecular weight, than POLY_(A). Preferably, themolecular weight of POLY_(B) is at most half that of POLY_(A) (excludingfunctional groups).

It can be seen that oligomeric and even monomeric lengths of POLY_(B)are included. In selected embodiments, POLY_(B) has at least two, or atleast three, monomeric units. POLY_(B) may have exactly two or exactlythree monomeric units. In further embodiments, the segment POLY_(B) hasfrom two to about 15 monomeric units. Preferably, when POLY_(B) consistsof one or two monomeric PEG units, in a segmented PEG polymer wherePOLY_(A) is mPEG, the functional group Y is not a maleimide group.

The molecular weight of the POLY_(B) segment is typically in the rangeof from about 100 Da to about 10,000 Da, and more typically from about100 Da to about 5000 Da, depending on the molecular weight of theindividual monomeric units. In selected embodiments, POLY_(B) has 2, 3,4, 5, 10, 15, 25, 35, 45, 75 or 100 monomeric units. These rangescorrespond, for example, to poly(ethylene glycol) segments havingmolecular weights of about 88, 132, 176, 220, 440, 660, 1100, 1540,1980, 3300, and 4400 Daltons, respectively. In other selectedembodiments, a PEG POLY_(B) segment has a molecular weight of about 100,500, 1000, 1500, 2000, 3000, or 5000. Selected ranges of molecularweight include from 44 to about 4400, 44 to about 1500, 220 to about2000, and about 2000 to about 3300. Numerous low molecular weight PEGs,having molecular weights of, for example, 200, 300, 400, 600, 900, 1000,2000, 3200, 3400, and 5000, are commonly available commercially.

POLY_(A) may be a “high” molecular weight segment, i.e. having more than200 monomeric repeating units, or a “medium” molecular weight segment,i.e. having 4 to about 200 monomeric repeating units. In selectedembodiments, POLY_(A) is a “medium molecular weight” segment having atleast 10, 20, 25, 50, 100, 125, 150, or 175 monomeric units, but at most200 monomeric units. These values correspond, for example, topoly(ethylene glycol) segments having molecular weights of at leastabout 440, 880, 1100, 2200, 4400, 5500, 6600, and 7700 Daltons,respectively, up to about 8800 Daltons. Typical ranges include about 25,about 50, about 100, or about 150-200 monomeric units.

In other selected embodiments, a “high molecular weight” POLY_(A)segment has at least 200, 250, 500, 1000, 1500, or 2000 or moremonomeric units. These ranges correspond, for example, to poly(ethyleneglycol) segments having molecular weights of at least about 8800,11,000, 22,000, 44,000, 66,000, or 88,000 or more Daltons, respectively.

In selected embodiments of segmented PEG-based polymers, where each ofPOLY_(A) and POLY_(B) is a poly(ethylene glycol) segment, POLY_(A) has amedium molecular weight range selected from about 200-5000, about500-2000, and about 1000-1500. In other embodiments of PEG-basedsegmented polymers, POLY_(A) has a high molecular weight range selectedfrom about 8800 to about 20000, about 15000 to about 50000, and about20000 to about 90000. Commercially available PEGs include those having anominal molecular weight of 10,000, 12,000, 15,000, 18,000, and 20,000,30,000, 40,000 and above. Branched PEGs are readily available at highermolecular weights.

B. Composition

Various monomers are available for preparing the water soluble segmentedpolymers of the invention. The segments of the polymer (i.e. POLY_(A)and POLY_(B)) can be formed from a single monomer (homopolymeric) or twoor three monomers (copolymeric). Preferably, each such segment is acopolymer of two monomers or, more preferably, a homopolymer. Themonomer(s) employed result in a segmented polymer that is water solubleas defined herein; that is, >95% water soluble, preferably >99% watersoluble, in water at room temperature at physiological pH (about7.2-7.6).

Accordingly, each of the segments designated POLY_(A) and POLY_(B)independently comprises up to three different monomers selected from thegroup consisting of: alkylene glycol, such as ethylene glycol orpropylene glycol; olefinic alcohol, such as vinyl alcohol, 1-propenol or2-propenol; vinyl pyrrolidone; hydroxyalkyl methacrylamide orhydroxyalkyl methacrylate, where alkyl is preferably methyl; saccharide;α-hydroxy acid, such as lactic acid or glycolic acid; phosphazene,oxazoline, and N-acryloylmorpholine. Preferred monomer types includealkylene glycol, olefinic alcohol, hydroxyalkyl methacrylamide ormethacrylate, N-acryloylmorpholine, and α-hydroxy acid. Preferably, eachsegment is, independently, a copolymer of two monomers selected fromthis group, or, more preferably, a homopolymer of one monomer selectedfrom this group. Typically, POLY_(A) and POLY_(B) will include the samemonomer(s).

The two monomers in a copolymer may be of the same monomer type, forexample, two alkylene glycols, such as ethylene glycol and propyleneglycol. In selected embodiments, each of POLY_(A) and POLY_(B) isindependently a poly(alkylene glycol). For example, each of POLY_(A) andPOLY_(B) may be selected from poly(ethylene glycol) and an ethyleneglycol/propylene glycol copolymer.

The monomers in a copolymer may also be of different monomer types. Forexample, the segmented water soluble polymer can be prepared with one ormore weak or degradable linkages in the polymer backbone, such as apoly(alkylene glycol) having periodic ester linkages in the polymerbackbone.

In preferred embodiments, each of POLY_(A) and POLY_(B) is apoly(ethylene glycol). PEG polymers are typically clear, colorless,odorless, soluble in water, stable to heat, inert to many chemicalagents, and non-toxic, and do not hydrolyze or deteriorate (unlessspecifically designed to do so). Poly(ethylene glycol) is highlybiocompatible and substantially non-immunogenic. When conjugated to apharmacologically active agent, the PEG molecule tends to mask the agentand can reduce or eliminate any immune response to the agent. PEGconjugates tend not to produce a substantial immune response or causeclotting or other undesirable effects.

The unconjugated terminus of the water soluble polymer, typically aterminus of POLY_(A), is generally capped to render it unreactive. Thecapping group may be, for example, C₁-C₂₀ alkoxy or aryloxy, morepreferably C₁-C₁₀ alkoxy or aryloxy. In further selected embodiments, Ris C₁-C₅ alkoxy or benzyloxy. A common is example is amethoxy-terminated PEG, designated mPEG.

The capping group may also be a phospholipid. A preferred phospholipidis a dialkyl phosphatidylethanolamine, such as distearoylphosphatidylethanolamine (DSPE; see structure below). Other suitablephospholipids include, for example, phosphatidyl serines, phosphatidylglycerols, phosphatidyl inositols, and phosphatidyl cholines, all ofwhich are well known in the art and available commercially. A reactivegroup on the phosphate head group can be used to link the lipid to a PEGchain; for example, the terminal amine in DSPE can be linked to PEG viaa carbamate linkage.

C. Linkages

The linkage X between the polymer segments is typically formed byreaction of a functional group on a terminus of POLY_(A) with acorresponding functional group on a terminus of POLY_(B). For example,an amino group on POLY_(A) may be reacted with an activated carboxylicacid derivative on POLY_(B), or vice versa, to produce an amide linkage.Alternatively, reaction of an amine with an activated carbonate (e.g.succinimidyl or benzotriazyl carbonate) forms a carbamate linkage, andreaction of an amine with an isocyanate (R—N═C═O) forms a urea linkage(R—NH—(C═O)—NH—R′). In selected embodiments, X comprises an amide, acarbamate, a carbonate, an ester, a urea, an amine, a thioether, or adisulfide. These linkages and methods of forming them are well known inthe art. Functional groups such as those discussed in Section D below,and illustrated in the working examples, are typically used for formingthe linkages. The linkage may also comprise (or be adjacent to orflanked by) spacer groups, as described further below.

Typically, the ten Unus of POLY_(A) not bearing a functional group iscapped to render it unreactive. As described above, suitable cappinggroups include alkoxy groups, aryloxy groups, and phospholipids, such asDSPE. When POLY_(B) includes a further functional group (Y) forformation of a conjugate, the group Y is either selected such that it isunreactive under the conditions of formation of the linkage X, or it isprotected during the formation of the linkage X.

In the segmented polymer compositions of the invention, the linkage X isdistinct from the monomeric units of the polymer segments and thelinkages between the monomers, such that there is a clear delineationbetween the structure of the linkage and the structure of the polymersegments. For example, in a segmented polymer in which POLY_(A) and/orPOLY_(B) is a PEG copolymer having periodic carboxylic ester linkagesfor the purpose of biodegradability, the linkage X between POLY_(A) andPOLY_(B) would not be such a carboxylic ester linkage (and clearly wouldnot be a —CH₂—CH₂O— linkage).

D. Functional Groups for Conjugation

The segmented polymers include a functional group, Y, attached toPOLY_(A) and/or POLY_(B), typically on POLY_(B), which is useful forRuining a conjugate of the polymer, e.g., with a pharmacologicallyactive agent, surface, solid support, or the like, as described ingreater detail below. The functional group typically comprises anelectrophilic or nucleophilic group that provides for covalentattachment of a desired agent to the segmented polymer. Examples ofnucleophilic groups include hydroxyl, amine, hydrazine (—NHNH₂),hydrazide (—C(O)NHNH₂), and thiol. Preferred nucleophiles include amine,hydrazine, hydrazide, and thiol, particularly amine.

Examples of electrophilic functional groups include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, halosilane, andphosphoramidate. More specific examples of these groups includesuccinimidyl ester or carbonate, imidazoyl ester or carbonate,benzotriazole ester or carbonate, vinyl sulfone, chloroethylsulfone,vinylpyridine, pyridyl disulfide, iodoacetamide, glyoxal, dione,mesylate, tosylate, and tresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, etc., as well as hydrates orprotected derivatives of any of the above moieties (e.g. aldehydehydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal). Another useful conjugation reagent is2-thiazolidine thione.

As noted above, an “activated derivative” of a carboxylic acid refers toa carboxylic acid derivative which reacts readily with nucleophiles,generally much more readily than the underivatized carboxylic acid.Activated carboxylic acids include, for example, acid halides (such asacid chlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g. hydroxy, thio, or amino groups, to produce various bond types. Forexample, carboxylic acids and activated derivatives thereof, whichinclude orthoesters, succinimidyl esters, imidazolyl esters, andbenzotriazole esters, react with the above types of nucleophiles to formesters, thioesters, and amides, respectively, of which amides are themost hydrolytically stable. Carbonates, including succinimidyl,imidazolyl, and benzotriazole carbonates, will react with hydroxyl oramino groups to form further carbonates or carbamates, respectively.Isocyanates (R—N═C═O) react with hydroxyl or amino groups to form,respectively, carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages.Phosphoramidites can be reacted with hydroxyl reagents, followed byoxidation, to form phosphate esters (as in conventional oligonucleotidesynthesis).

Aldehydes, ketones, glyoxals, diones and their hydrates or alcoholadducts (i.e. aldehyde hydrate, hemiacetal, acetal, ketone hydrate,hemiketal, and ketal) are preferably reacted with amines, followed byreduction of the resulting imine, if desired, to give an amine linkage(reductive amination). Alternatively, these groups can be reacted withhydroxyl containing groups, to form further acetals, ketals, etc. Inthese cases, the linkages formed are subject to hydrolytic degradation,which may be desirable, as discussed further below.

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such a thiols, can be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups which can be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Silanes,including halosilanes and alkoxysilanes, react with hydroxy- oroxide-containing compounds, or surfaces such as glass, to formsiloxanes.

The use of several of these groups is described in the followingrepresentative references: N-succinimidyl carbonate (U.S. Pat. Nos.5,281,698 and 5,468,478), N-succinimidyl succinate (Abuchowski et al.,Cancer Biochem. Biophys. 7:175, 1984), N-succinimidyl ester (U.S. Pat.No. 4,670,417), N-succinimidyl propionate (U.S. Pat. No. 5,672,662),N-succinimidyl butanoate (U.S. Pat. No. 5,672,662), benzotriazolecarbonate (U.S. Pat. No. 5,650,234), propionaldehyde (U.S. Pat. No.5,824,784) and acetaldehyde, which are typically protected as thediethyl acetal (U.S. Pat. No. 5,990,237), glycidyl ether (Elling et al.,Biotech. Appl. Biochem. 13:354, 1991), imidazolyl ester (Tondelli etal., J. Controlled Release 1:251, 1985), p-nitrophenyl carbonate(Sartore et al., Appl. Biochem. Biotech. 27:45, 1991), maleimide (Kogan,Synthetic Comm. 22:2417, 1992), orthopyridyl disulfide (Woghiren et al.,Bioconj. Chem. 4:314, 1993), acrylate (Sawhney et al., Macromolecules26:581, 1993), vinyl sulfone (U.S. Pat. No. 5,900,461), and2-thiazolidine thione (Greenwald et al., U.S. Pat. No. 5,349,001).

E. Spacers

One or more spacer groups may be employed to link the functional groupY, or the linkage X, to a terminus or branch point of segment POLY_(A)or POLY_(B). Preferably, the atoms making up the spacer moiety comprisesome combination of carbon, oxygen, hydrogen, nitrogen, and, lessfrequently, sulfur atoms. The spacer generally does not include bondsthat are reactive under physiological conditions, unless it is desiredthat the spacer be hydrolytically or enzymatically cleavable.Accordingly, the spacer(s) may be hydrolytically stable, including, forexample, bonds such as alkyl, ether, keto, amide, urea, or sulfide, orthey may include a physiologically hydrolyzable or enzymaticallydegradable linkage, such as an ester, carbonate, carbamate, ordisulfide.

Preferred spacer groups include hydrocarbon spacers, e.g. linear orbranched divalent alkyl chains having 4 to about 24, preferably 4 toabout 12, more preferably 4 to 8, carbon atoms, e.g. as tetramethylene,pentamethylene, or hexamethylene. Other hydrocarbon-based spacersinclude bivalent cycloalkyl groups, preferably C₃-C₈ cycloalkyl, such ascyclopropadiyl, cyclobutadiyl, cyclopentadiyl, cyclohexadiyl, andcycloheptadiyl. The cycloalkyl group can be substituted with one or morealkyl groups, preferably C₁-C₆ alkyl groups.

III. Structural Variants

The segmented polymer and/or the individual segments, POLY_(A) andPOLY_(B), can have various architectural forms, e.g. linear, branched,forked, multi-armed, or dendrimeric. Some of these forms, such as forkedor multiarmed, typically contain two or more functional groups forconjugation. Such multifunctional polymers can be used to conjugatemultiple therapeutic molecules and/or targeting agents to a singlecarrier. They are also useful for linking macromolecules to surfaces,for use in assays or biosensors, for example. The water solublesegmented polymers can also be used to form crosslinked or uncrosslinkedhydrogels, which are particularly useful for drug delivery devices andbiocompatible wound coverings.

A. Linear Polymers

A linear polymer of the invention has the general structure:

POLY_(A)-X-POLY_(B)-Y   (Ia)

or

POLY_(B)-X-POLY_(A)-Y   (Ib)

where structure Ia is generally preferred. Linear concatamers, e.g.containing multiple linked POLY_(A) and/or POLY_(B) segments, may alsobe provided.

In further selected embodiments, a linear segmented PEG polymer isprovided, having the structure:

R—(CH₂CH₂O)_(n)—CH₂CH₂—X—(CH₂CH₂O)_(m)—CH₂CH₂—Y   (Ic)

where:

R is a functional group or, preferably, a capping group;

n is at least 4 and at most about 2000,

m is between 1 and about 120, where m<n,

X is a linking group, as described herein, and

Y is a functional group, as described herein, which may be the same ordifferent from R when R is a functional group. In selected embodiments,n is at most about 1000, at most about 500, or at most about 200.

The capping group may be, for example, C₁-C₂₀ alkoxy or aryloxy, morepreferably C₁-C₁₀ alkoxy or aryloxy. In further selected embodiments, Ris C₁-C₅ alkoxy or benzyloxy.

The capping group may also be a phospholipid. A preferred phospholipidis a dialkyl phosphatidylethanolamine, such as distearoylphosphatidylethanolamine (DSPE), where the terminal amine of thephosphate head group can be used to link the lipid to the PEG chain via,for example, a carbamate linkage. Other suitable phospholipids include,for example, phosphatidyl cholines and phosphatidyl inositols.

Several exemplary linear segmented PEGs are shown in Examples 4, 6-7,9-14, 16-17 and 20 below. A particularly preferred class of compound isillustrated in Examples 14A-C. These compounds have the structure:

CH₃O—(CH₂CH₂O)_(n)—(CO)NH—CH₂CH₂OCH₂CH₂OCH₂CH₂NH₂

The molecular weight of the segmented polymer is dependent on the sizeof the POLY_(A) segment (CH₂CH₂O)_(n) and is preferably selected fromabout 1000, about 5000, about 10000, and about 20000.

B. Branched Polymers

A “branched” polymer of the invention, in one aspect, refers to anysegmented polymer as described herein in which POLY_(A) and/or POLY_(B),preferably POLY_(A), is branched. The polymer segment may include branchpoints within its monomeric units, e.g. where some percentage ofmonomers is trifunctional or greater. The polymer segment may alsocomprise multiple polymer arms connected to a common linking group.

Certain branched polymers of the invention can be representedschematically as follows:

(POLY_(A)-X-POLY_(B))_(p)-L-Y   (IIa)

or

(POLY_(A)-X)_(p)-L-POLY_(B)-Y   (IIb)

or

(POLY_(A))_(p)-L-X-POLY_(B)-Y   (IIc)

where POLY_(A), POLY_(B), X, and Y are as defined above; L represents abranched spacer group; and p is ≧2, preferably 2 to about 100, morepreferably 2 to about 10. In one embodiment, p is 2.

For example, in structure Ia above, multiple polymer arms, eachcomprising POLY_(A) linked to POLY_(B), are attached, via a branchedspacer group, to a single functional group Y. Alternatively, POLY_(A)can be a branched polymer segment, having 2 or more polymer arms, whichis attached via a covalent linkage to a single POLY_(B)-Y segment, as instructure IIc or IIb above.

Preferably, the branch point in the branched spacer group L comprises acarbon atom (—CH<), though it may also comprise a nitrogen atom (—N<).The branch point is linked to adjacent moieties, such as X, Y or apolymer segment, directly or by chains of atoms of defined length. Eachchain of atoms may comprise, for example, alkyl, ether, ester, or amidelinkages, or combinations thereof.

Illustrative branched POLY_(A) segments include those described in U.S.Pat. No. 5,932,462, the content of which is incorporated herein byreference. Generally speaking, branched POLY_(A) segments of this sortare characterized by the structure:

where POLY_(A) is as defined herein, and P and Q are hydrolyticallystable linkage fragments joining the polymer arms, POLY_(A), to thecentral carbon atom, C. R is typically H or methyl, but may comprise alinkage fragment joined to an additional polymer arm, POLY_(A).

In selected embodiments of structures IIa-c above, the POLY_(A) segmentis a PEG segment (PEG_(A)) comprising at least 4 and at most about 2000—CH₂CH₂O— monomeric units; X is selected from an amide, a carbamate, acarbonate, an ester, a urea, an amine, a thioether, and a disulfide; andthe POLY_(B) segment is a PEG segment (PEG_(B)) comprising at least 1,preferably at least 2, and at most about 120 —CH₂CH₂O— monomeric units,with the proviso that PEG_(B) has a lower molecular weight than that ofPEG_(A). Preferred ranges of p include 1-100, 2-20, and 2-10.

In these structures and in others described herein, PEG_(A) representsan embodiment of POLY_(A), and preferably has a size and molecularweight range as described for POLY_(A) above. Examples of molecularweight ranges include about 10000-40000, or, for medium molecular weightembodiments, about 200-1000 or 1000-5000. PEG_(A) preferably comprisesan end-capping group, such as alkoxy or a phospholipid. In selectedembodiments, X comprises a carbamate, an amide, a carbonate, an ester, aurea, an amine, a thioether, or a disulfide. and Y comprises an amine orprotected amine. Preferably, X comprises a carbamate, an amide, a urea,or a carbonate.

One example of a branched PEG segment which could be represented by(POLY_(A)-X)₂-L- above is a PEG-disubstituted lysine having thestructure:

The preparation of an exemplary branched polymer of the invention, inaccordance with the structure (POLY_(A))_(p)-L-X-POLY_(B)-Y (IIc)presented above, is provided in Examples 18 and 19, where a branchedPOLY_(A) segment (each branch comprising 20 kDa PEG) is linked to afunctionalized POLY_(B) segment (3400 MW PEG), having an aldehydefunctional group at the terminus, via an amide linkage:

C. Forked Polymers

A “forked” polymer of the invention refers to a single segmented polymer(which can itself be, for example, linear or branched) which is linkedto two functional groups, or, in a conjugate, to two pharmacologicallyactive agents. See e.g. PCT Pubn. No. WO 99/45964. Certain forkedpolymers of the invention can be represented schematically as follows:

POLY_(A)-L-(X-POLY_(B)-Y)₉   (IIIa)

or

POLY_(A)-X-L-(POLY_(B)-Y)₉   (IIIb)

or

POLY_(A)-X-POLY_(B)-L-(Y)_(q)   (IIIc)

where POLY_(A), POLY_(B), X, and Y are as defined above; L represents abranched spacer group; and q is ≧2, preferably 2 to about 100, morepreferably 2 to about 10. In one embodiment, q is 2. Preferably, thebranch point in the spacer group comprises a carbon atom (>CH—), thoughit may also comprise a nitrogen atom (>N—).

An exemplary forked polymer that may be represented byPOLY_(A)-X-L-(POLY_(B)-Y)₉ (structure IIIb above) possesses thestructure:

where W and W′ are first and second tethering groups that can be thesame or different, and comprise POLY_(B) segments, connected to thebranching atom, —CH, optionally via intervening spacer groups. In aparticular embodiment, W and W′ are identical.

A polymer of the invention may be forked as well as branched; forexample, it may have the schematic structure(POLY_(A))₂-L-X-L′-(POLY_(B)-Y)₂. L and L′ may be the same or different.An exemplary structure of this type, having two maleimide functionalgroups, is:

Preferably, in a forked PEG or a forked/branched PEG as illustratedabove, where PEG_(A) is mPEG and the functional group Y is a maleimidegroup, the segment PEG_(B) has a molecular weight of at least 130,preferably at least 200, and more preferably at least 300.Alternatively, PEG_(B) includes at least three, preferably at leastfour, and more preferably at least 10 monomeric PEG units.

Either POLY_(A) or POLY_(B) may also include pendant functional groupscovalently attached along the length of the PEG backbone. The pendantreactive groups can be attached to the PEG backbone directly or througha spacer group, such as an alkylene group.

D. Multiarmed and Dendrimeric Polymers

A “multiarmed” polymer of the invention comprises three or morewater-soluble polymer chains attached to a central core structure, andcan be represented by the general structure:

M-{POLY_(A)-X-(POLY_(B)-Y)_(q)}_(x)   (IV)

where M is a core structure, such as a polyol, having several linkagepoints for attachment of POLY_(A), and x represents the number of“arms”.

The value of x is less than or equal to the valence of the multivalentcore structure, M. Typically, the value of x is equal to the valence ofthe core structure, M.

The value of x is typically 3 to about 100, more typically 3 to about20. For example, a polyol core structure can generally provide from 3 toabout 100 available hydroxy groups, and typically provides 3 to about20, so that the branched polymer structure has from about 3 to about100, more typically 3 to about 20, “arms”. The core structure M ispreferably selected from a polyol, a polyamine, and an amino acid whoseside chain bears a functional group, and more preferably selected from apolyol and a polyamine. Types of suitable core structures are discussedfurther below.

In selected embodiments of structure (IV), q is 1 or 2; that is, each“arm” represents a linear polymer (q=1) or a “forked” polymer (q=2). Infurther embodiments, x is 3 to 20, or 3 to 10. Generally, q is 1, suchthat each POLY_(A) is linked to a single POLY_(B). However, q can begreater than 1, and POLY_(B) can include more than one Y or apolyfunctional Y, as noted above. Where q is >1 or POLY_(B) includesmore than one Y, branched spacer groups, as represented by L above, areincorporated as appropriate. Generally, POLY_(B) includes a single groupY.

The multiarmed polymer may also include some POLY_(A) segments which arenot further substituted. Accordingly, structure (IV) can represent abranched or dendritic polymer segment in which each of a plurality ofPOLY_(A) polymer arms (where the plurality is equal to or less than q)is covalently attached to a POLY_(B)-Y segment via a covalent linkage,X. The remaining POLY_(A) polymer arms may be uncapped (i.e. hydroxylterminated) or capped with a capping group such as described above,preferably an alkoxy group.

In selected embodiments, the multiarmed polymer is a PEG polymer, e.g.:

M-{PEG_(A)-X-(PEG_(B)-Y)_(q)}_(x)   (IV′)

where PEG_(A) represents poly(ethylene glycol) comprising at least 4 andat most about 2000 —CH₂CH₂O— monomeric units, and PEG_(B) representspoly(ethylene glycol) comprising at least 1 and at most about 120—CH₂CH₂O— monomeric units, with the proviso that PEG_(B) has a lowermolecular weight than that of PEG_(A). As above, the multiarmed polymermay also include some PEG_(A) segments which are not furthersubstituted. Accordingly, structure (IV′) can represent a branched ordendritic polymer segment in which each of a plurality of PEG_(A)polymer arms (where the plurality is equal to or less than q) iscovalently attached to a PEG_(B)-Y segment via a covalent linkage, X.The remaining PEG_(A) arms may be uncapped (i.e. hydroxyl terminated) orcapped with a capping group such as described above, preferably analkoxy group.

Since PEG_(A) and PEG_(B) are embodiments of POLY_(A) and POLY_(B),respectively, their preferred size and molecular weight rangescorrespond to those described for POLY_(A) and POLY_(B) above. Inselected embodiments, X comprises a carbamate, an amide, a carbonate, anester, a urea, an amine, a thioether, or a disulfide. Preferably, Xcomprises a carbamate, an amide, a urea, or a carbonate.

Dendrimeric polymers of the invention comprise multiple (e.g., 3 to 50)water-soluble segmented polymers connected to a core structure. Suchforms can be distinguished from “multi-armed” forms, where the “arms”are generally linear or moderately branched, in that dendrimericpolymers are very highly branched, with branching increasing with thedistance from the core structure. Generally, each of a plurality (orall) of the arms will be linked to a POLY_(B) segment and thence to afunctional group Y.

D1. Core Structures

The core structure in multiarmed and dendrimeric polymers can be anygroup containing multiple linkage points for attachment of the polymerchains. Examples include polyols, polyamines, and amino acids havingfunctional side chains. Of these, polyols and polyamines are preferred.

Polyols that are suitable for use as the polymer core are nearlylimitless. Aliphatic polyols having from 1 to 10 carbon atoms and from 1to 10 hydroxyl groups may be used, including ethylene glycol, otheralkane diols, alkylidene alkyl diols, alkyl cycloalkane diols, alkanepolyols such as glycerol or pentaerythritol, cycloalkylidene diols suchas 1,5-decalindiol or 4,8-bis(hydroxymethyl)tricyclodecane, and thelike. In one embodiment, branched polyols of the formHO—(CH(OH)CH₂O)_(n)H, prepared by condensation of glycerol, areemployed.

Cycloaliphatic polyols may also be employed, including straight chainedor closed-ring sugars and sugar alcohols, such as mannitol, sorbitol,inositol, xylitol, quebrachitol, threitol, arabitol, erythritol,adonitol, dulcitol, facose, ribose, arabinose, xylose, lyxose, rhamnose,galactose, glucose, fructose, sorbose, mannose, pyranose, altrose,talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like.In general, any carbohydrate core, preferably a mono- or disaccharide,can be used, after reduction of aldehydes to hydroxyl groups, ifdesired.

Aromatic polyols may also be used as cores, particularly when a morehydrophobic core structure is desired. Suitable aromatic polyols include1,1,1-tris(4′-hydroxyphenyl)alkanes, such as1,1,1-tris(4-hydroxyphenyl)ethane, (1,3-adamantanediy)diphenol,2,6-bis(hydroxyalkyl)cresols,2,2′alkylene-bis(6-t-butyl-4-alkylphenols),2,2′-alkylene-bis(t-butylphenols), catechol, alkylcatechols, pyrogallol,fluoroglycinol, 1,2,4-benzenetriol, resorcinol, alkylresorcinols,dialkylresorcinols, orcinol monohydrate, olivetol, hydroquinone,alkylhydroquinones, 1,1-bi-2-naphthol, phenyl hydroquinones,dihydroxynaphthalenes, 4,4′-(9-fluorenylidene)-diphenol, anthrarobin,dithranol, bis(hydroxyphenyl)methane biphenols, dialkylstilbesterols,bis(hydroxyphenyl)alkanes, bisphenol-A and derivatives thereof,meso-hexesterol, nordihydroguaiaretic acid, calixarenes and derivativesthereof, tannic acid, and the like.

Other core polyols that may be used include crown ethers, cyclodextrins,dextrins, and other carbohydrates such as starches and amylose. See alsoU.S. Pat. No. 5,932,462 (Harris et al., 1999), which is incorporated byreference herein in its entirety.

A large variety of polyamines are also available for use as corestructures, including aliphatic diamines, triamines, tetramines, etc.Well known examples, some of which are naturally occurring, includeα,ω-aliphatic diamines, e.g. 1,6-hexanediamine. Other diamines havingdiverse structures are commercially available.; for example,3-dimethylamino propylamine, 3-diethylaminopropylamine, N,N-dimethyldipropylenetriamine, N,N′-di-t-butyl ethylenediamine, andbis(aminomethyl)tricyclododecane are available from Celanese Chemicals.

Aliphatic polyamines which are naturally occurring include spermidine(an aliphatic triamine) and spermidine (an aliphatic tetramine). Alsouseful as core structures are aliphatic polyamines, some of whichinclude unsaturation and intervening cycloalkyl or aryl groups, reportedfor use as antiinfective agents in C. J. Bacchi et al., AntimicrobialAgents and Chemotherapy 46(1):55-61 (January 2002). These includetetramines of the form EtNH-R¹—NH—CH₂—R²—CH₂—NH—R₁—NHEt, where each R¹is n-butyl or cis-2-butenyl, and R² is selected from ethyl, cis-ethenyl,ethynyl, cyclopropyl (cis or trans), cyclobutyl (cis or trans), andortho-phenyl. Also included are pentamines of the formEt-(NH—R¹)_(4—)NHR³, where R¹ is again n-butyl or cis-2-butenyl and R³is ethyl, 2-hydroxyethyl or 2-aminoethyl. Also included are higheroligoamines of the faunEt-(NHCH₂CH₂CH₂CH₂)₃₋₆—NH—R¹—(NHCH₂CH₂CH₂CH₂)₃₋₆—NHEt, where R¹ is againn-butyl or cis-2-butenyl.

E. Hydrogels

The water soluble polymers of the invention can also be used to formhydrogels, particularly crosslinked hydrogels (see U.S. Pat. No.6,413,507, Bentley et al., 2002, which is incorporated herein byreference). Such gels, and conjugates based on these gels, areparticularly useful for biocompatible controlled release dosage formsand wound dressings. Aqueous hydrogels can be used in various biomedicalapplications, such as soft contact lenses, wound management, drugdelivery, and implants for tissue replacement and augmentation.

A crosslinked hydrogel of the invention comprises, in one embodiment, awater soluble segmented polymer as described herein, bonded to acrosslinking agent through a hydrolyzable linkage. The polymer has afunctional group Y, such as an amino or hydroxyl group, available toreact with the crosslinking agent to form a hydrolyzable linkage, suchas a carbamate, carbonate or ester linkage. Preferably, the polymer hasat least two such functional groups. In selected embodiments, thepolymer comprises PEG segments. The functional groups are typically onPOLY_(B), but they may be present on POLY_(A) or on both segments.

Accordingly, the polymer used to form the hydrogel can take one of thefollowing forms:

Y-POLY_(B)-X-POLY_(A)-X-POLY_(B)-Y

or

M{POLY_(A)-X-POLY_(B)-Y}_(x)

where X, Y, M, and x are as defined above.

The polymer is reacted with a crosslinking moiety which includes atleast two groups effective to react with Y to fotin a covalent linkage.

The pharmacologically active agents to be delivered can be part of thecrosslinking moiety of the hydrogel, or they can be incorporated in“prodrug” form by covalent linkage to the polymer backbone or to thecrosslinking moiety. Agents to be delivered can also be loaded into thehydrogel during its synthesis, or afterwards, e.g., by diffusion intothe cavity or matrix of the hydrogel, without being covalently bonded tothe hydrogel structure.

The hydrogel can be used as a carrier for delivery of pharmacologicallyactive agents, e.g. drugs, nutrients or labeling agents for imaginganalysis, and for other suitable biomedical applications. For example,the hydrogel can carry therapeutic drugs and can be implanted orinjected in the target area of the body.

Drugs which are physically trapped in the gel are released by diffusionas the gel degrades. Agents which are covalently bound throughhydrolyzable linkages are released at a controllable rate as theselinkages degrade. Because the hydrogel is characterized by theinterconnection of a large number of hydrolytically degradable linkages,the degradation or breakdown of the hydrogel in the body is typicallygradual in nature. Thus, it is particularly useful for sustained releaseof an agent in the body.

IV. Preparation of the Segmented Polymers

The invention includes methods of preparing the segmented water-solublepolymers. In general, a first water soluble polymer segment, such asthat designated herein as POLY_(A), having at least one functionalgroup, Z, is provided, and is reacted with a second water solublesegment, such as that designated herein as POLY_(B), having at least onefunctional group, Y′, thereby covalently bonding the first and secondsegments by reaction of Z with Y′. Either POLY_(A) or POLY_(B),preferably POLY_(B), comprises an additional functional group, Y, thatis not readily reactive with either Z or Y′ under the conditions ofreaction of those two groups.

In general, the method and resulting functionalized polymer can berepresented by:

POLY_(A)-Z+Y′-POLY_(B)-Y→POLY_(A)-X-POLY_(B)-Y

In selected embodiments, each of the first and second segments is apoly(alkylene) glycol), preferably a PEG. Typically, POLY_(A) includes acapping group, such as an alkoxy or aryloxy group, at one terminus, andthe functional group Z at the other terminus, and POLY_(B) isheterobifunctional, having Y and Y′ at its respective termini. When Yand Y′ are different, a protecting strategy may be used that renders Y′reactive while Y remains unreacted.

The unreacting functional group Y can comprise any of the functionalgroups described herein, typically in protected form, as long as it isunreactive under the conditions of reaction of Z and Y′. These groupsinclude, for example, nucleophiles such as hydroxyl, amino, hydrazine,hydrazide, or thio, and electrophiles such as carboxylic acids;carboxylic esters or carbonates, including imide esters or carbonates,imidazoyl ester or carbonate, and benzotriazole ester or carbonate;orthoesters; isocyanates; isothiocyanates; aldehydes, including glyoxal;ketones, including diones; thiones; alkenyl, including vinyl pyridineand vinyl sulfone; acrylate; methacrylate; acrylamide; sulfone;maleimide; disulfide, including orthopyridyl disulfide; halo, e.g.chloroethyl sulfone and iodoacetamide; epoxy, including glycidyl ether;sulfonate, including tosylate, mesylate, and tresylate; thiosulfonate;silane; alkoxysilane; halosilane; and phosphoramidate. These groups areprovided in protected form if necessary. In a preferred embodiment, Y isa protected amino group, such that the segmented polymer has an aminogroup available for conjugation.

Generally, one of Z and Y′ is a nucleophile, e.g. amine, hydrazine,hydrazide, thiol, or hydroxyl, and the other is an electrophilic group,e.g. N-succinimidyl carbonate, succinimidyl ester, benzotriazolecarbonate, isocyanate, glycidyl ether, imidazolyl ester, p-nitrophenylcarbonate, aldehyde, maleimide, ortho-pyridyl disulfide, acrylate, orvinyl sulfone. See, for example, the discussion of functional groupsprovided above. The nucleophilic group may be present on either thePOLY_(A) or the POLY_(B) segment, with a corresponding electrophilicgroup present on the POLY_(B) or POLY_(A) segment, respectively. Any ofthese groups can be provided in protected form on the starting polymersegments and deprotected prior to reaction or in situ. See, for example,the working Examples provided below. Typical reaction schemes includereaction of an amine, on either POLY_(A) or POLY_(B), with an activatedester, carbonate, or isocyanate, to form an amine, carbamate, or urealinkage, respectively. One preferred strategy, as illustrated in theworking Examples below, employs the reaction of either an NHS ester or abenzotriazolyl carbonate (as group Z on POLY_(A)) with an amine (asgroup Y′ on POLY_(B)) to form a carbamate or amide as the linkage X.Embodiments of Y illustrated in these Examples include thiosulfonate,carboxylic acid, carboxylic ester, amine, aldehyde or protected aldehyde(acetal), and maleimide.

These strategies can be readily adapted for preparation of otherstructural types, such as branched, forked, or multi-arm polymers. Forexample, in a typical preparation of a branched polymer of the generalform:

(POLY_(A)-X-POLY_(B))_(q)-L-Y

such as described in Section IIIA above, a compound having the structure(Z′)_(q)-L-Y is reacted with Y′-POLY_(B)-Z″, where Z′ and Z″ areeffective to form linkages between each POLY_(B) and the spacer group,and Y and Y′ are unreactive under the conditions of this reaction, orare in protected form during the reaction. The resulting structure,(Y′-POLY_(B))_(q)-L-Y, is then reacted with POLY_(A)-Z as describedabove. Generally, q is 2.

In a typical preparation of a segmented multi-arm polymer, such asdescribed in Section IIIB above, a POLY_(A) segment, such as a PEGsegment having a molecular weight in the ranges described above forPOLY_(A), is attached to each linkage point on the core structure, e.g.each hydroxyl group of a polyol. These attachments may be via spacergroups or, more preferably, direct linkages. The free terminus of eachPOLY_(A) “arm” is then converted, if necessary, to a functional group Z,prior to combination with a POLY_(B) segment, such as a PEG segmenthaving a molecular weight in the ranges described above for POLY_(B),having a reactive group Y′. Preferably, the POLY_(B) segments, or atleast a plurality of the POLY_(B) segments, are heterobifunctional, asdescribed above, so that a functional group Y remains at the freeterminus of at least a plurality of the POLY_(B) segments in theresulting multi-aim segmented polymer. Again, the functional group(s) Ymay be in protected form.

As noted above, the segmented water soluble polymers can be preparedwith one or more weak or degradable linkages in the polymer backbone,such as a poly(alkylene glycol) having periodic ester linkages in thepolymer backbone. Other hydrolytically or enzymatically cleavablelinkages include carbonates, carbamates, and hydrazones. Some amides mayalso be degraded in vivo by peptidases. The introduction of one or moredegradable linkages into the polymer chain can provide additionalcontrol over the final desired pharmacological properties of theconjugate upon administration. For example, a large and relatively inertconjugate (i.e., having one or more high molecular weight PEG chainsattached thereto, for example, one or more PEG chains having a molecularweight greater than about 8,000, wherein the conjugate possesses littlebioactivity) may be administered, and upon hydrolysis in vivo generatesa bioactive conjugate possessing a smaller portion of the original PEGchain. In this way, the properties of the conjugate can be moreeffectively tailored to balance the bioactivity of the conjugate overtime. The polymers may also be used in the fabrication of in vivodegradable solid dosage fauns of the conjugated agent.

The linking reactions generally involve known reactions and are carriedout according to standard methods of organic synthesis, particularlypolymer modification methods. See, for example, the working examplesprovided herein. Suitable solvents typically include, but are notlimited to, tetrahydrofuran, dioxane, acetonitrile, methylene chloride,chloroform, dimethylformamide, dimethylsulfoxide, and, for less polarmaterials, benzene, toluene, xylenes, and petroleum ether.

As discussed above, it has been found that the modified and/orconjugated low molecular weight POLY_(B) segments undergo syntheticoperations such as dissolution, filtration, separation, and purificationwith more efficiency and higher yields than similarly modified and/orconjugated high molecular weight polymers. In general, it is easier toseparate mixtures of low molecular weight polymers into componentspecies than similar higher weight polymers. End group modification andconjugation is generally a multi-step process, with each step of thefunctionalization resulting in polymeric impurities. If purification isineffective, which can be the case when dealing with high molecularweight polymers, the impurities accumulate throughout functionalizationof the polymer to an unacceptable level. By first performingfunctionalization and purification processes on a low molecular weightpolymer and subsequently joining the purified low molecular weightpolymer with a high molecular weight polymer, in accordance with thisinvention, process steps involving the high molecular weight polymer areminimized, resulting in a high molecular weight functionalized orconjugated polymer derivative in overall desirable purity and yield.Accordingly, it is preferred, in synthesizing the subject polymers, thatthe step of linking the functionalized POLY_(B) segment(s) with thehigher molecular weight POLY_(A) segment is carried out near or at theend of the synthetic scheme

V. Preparation of Conjugates

In another embodiment of the invention, a method for preparing a polymerconjugate is provided. The method comprises the step of contacting awater soluble, segmented polymer as described herein, having at leastone functional group Y, with a pharmacologically active agent having acorresponding reactive group Z′, under suitable condition to produce acovalent linkage between Y and Z′. Y is frequently an electrophilicgroup and Z′ a nucleophilic group, though these roles may be reversed.Examples of suitable pharmacologically active agents are discussedbelow.

The specific conditions for effecting conjugation depend, in part, onthe functional group(s) Y present on the segmented polymer, themolecular weight of the polymer, and the reactive group(s) Z′ present inthe specific pharmacologically active agent, as well as the possiblepresence of additional reactive functional groups within the agent. Forany given polymer and pharmacologically active agent, however, suitablereaction conditions, including protection strategies if necessary, willbe known to one of ordinary skill in the art or can be identifiedthrough routine experimentation. Such conditions include pH,temperature, reagent concentration, and so forth.

For example, when the polymer contains an N-hydroxysuccinimide activeester (e.g., succinimidyl succinate, succinimidyl propionate, orsuccinimidyl butanoate), and the active agent contains an amine group(e.g., a terminal or side chain amine group on a polypeptide),conjugation can be effected at a pH of from about 7.5 to about 9.5 atroom temperature, to form an amide linkage. In analogous reactions,reaction of an amine with an activated carbonate (e.g. succinimidyl orbenzotriazyl carbonate) forms a carbamate linkage, and reaction of anamine with an isocyanate (R—N═C═O) forms a urea linkage(R—NH—(C═O)—NH—R′).

In further embodiments, when the polymer contains a vinyl sulfone ormaleimide (see e.g. U.S. Pat. No. 5,739,208; PCT Pubn. WO 01/62827), andthe pharmacologically active agent contains a thiol group (as in acysteine side chain on a polypeptide or protein), conjugation can beeffected at a pH of from about 7 to about 8.5 at room temperature, toform a sulfide (thioether) linkage.

Alternatively, when the functional group Y comprises an aldehyde, aketone, a hydrate thereof, an acetal or ketal, or a hemiacetal orhemiketal, and the pharmacologically active agent contains a primaryamine, conjugation can be effected by reductive amination, where theinitially formed imine bond is reduced to the amine with a suitablereducing agent such as NaCNBH₃. Such reactions can also be carried outwith the sulfur analogs of these functional groups.

Conversely, in the reactions described above, the polymer may containthe nucleophilic functional group, for reaction with an electrophilicgroup on the agent to be attached. As another example, an amine- orhydrazide-modified polymer can be used for conjugation to acarbohydrate-containing compound, such as a saccharide, a glycolipid ora glycoprotein, where the carbohydrate residue is first treated withperiodate to generate reactive aldehyde groups.

Other types of bonds formed by such conjugation are described above. Foradditional information concerning such conjugation reactions, referenceis made to Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, 1996.Several working examples of conjugation are also provided below in theExamples section.

The molecular weight and architecture of the water soluble polymer(including the number of available functional groups per a givenmolecular weight of polymer) can be selected based on the drug beingdelivered and condition being treated. For example, long circulatingtime is a known benefit of higher molecular weight polymer conjugates.However, in some cases, as in administering thrombolytics, longcirculating time may be undesirable. The use of a lower molecular weightpolymer can also reduce the overall weight of the composition for agiven dose of the active agent, and may be less likely to block anactive site on the agent.

Preferably, conjugates having a stable linkage (e.g., an amine or etherlinkage) between the polymer and the active agent will possess, inconjugate form, at least some degree of the bioactivity of theunmodified agent. The conjugates can also be designed as prodrugs, suchthat the covalent linkage between the segmented polymer and thepharmacologically active agent is degradable in vivo, to allow releaseof the agent. Exemplary degradable linkages include carboxylate esters,phosphate esters, thioesters, anhydrides, acetals, ketals, acyloxyalkylethers, imines, hydrazones (typically formed by reaction of a hydrazideand an aldehyde), disulfides, and orthoesters (formed by reactionbetween a formate and an alcohol). Linkages can often be tailored tocontrol the rate of cleavage, for example, by modifying the substitutionon an ester linkage. Other examples include linkages designed such thatcleavage is intramolecularly accelerated by a neighboring group, such asan amine, or the “benzyl elimination” system, involving cleavage of abenzyl disulfide (e.g. M. Wakselman, Nouveau J. de Chemie 7:439-447,1983).

VI. Pharmacologically Active Agents

The polymers of the invention can be conjugated to any pharmacologicallyactive agent which has a suitable functional group for attachment, forthe purpose of improving the pharmacological properties (such assolubility, biodistribution, rate of delivery, or metabolism) of theagent. The active agent is frequently a therapeutically active agent,i.e. a drug, but it may also include diagnostic agents, includingantibodies or other binding moieties, or dyes or other imagingmaterials, as well as targeting or delivery agents, e.g. molecules fortargeting specific receptors.

The active agent may fall into one of a number of structural classes,including but not limited to small molecules (including difficultlysoluble or insoluble small molecules), peptides, polypeptides, proteins,polysaccharides, steroids, nucleotides, oligonucleotides,polynucleotides, lipids, fats, electrolytes, and the like.

Biologically or therapeutically active agents to be conjugated tosegmented, water soluble polymer of the invention may be any one or moreof the following. Suitable agents may be selected from, for example,hypnotics and sedatives, psychic energizers, tranquilizers, respiratorydrugs, anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagnonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

Specific examples of active agents suitable for covalent attachment to apolymer of the invention include but are not limited to aspariginase,amdoxovir (DAPD), antide, becaplermin, calcitonins, cyanovirin,denileukin diftitox, erythropoietin (EPO), EPO agonists (e.g., peptidesfrom about 10-40 amino acids in length and comprising a particular coresequence as described in WO 96/40749), dornase alpha, erythropoiesisstimulating protein (NESP), coagulation factors such as Factor VIIa,Factor VIII, Factor IX, von Willebrand factor; ceredase, cerezyme,alpha-glucosidase, collagen, cyclosporin, alpha defensins, betadefensins, exedin-4, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), fibrinogen,filgrastim, growth hormones human growth hormone (hGH), growth hormonereleasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenicproteins such as bone morphogenic protein-2, bone morphogenic protein-6,OP-1; acidic fibroblast growth factor, basic fibroblast growth factor,CD-40 ligand, heparin, human serum albumin, low molecular weight heparin(LMWH), interferons such as interferon alpha, interferon beta,interferon gamma, interferon omega, interferon tau; interleukins andinterleukin receptors such as interleukin-1 receptor, interleukin-2,interluekin-2 fusion proteins, interleukin-1 receptor antagonist,interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6,interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17receptor; lactoferrin and lactoferrin fragments, luteinizing hormonereleasing hormone (LHRH), insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), influenza vaccine,insulin-like growth factor (IGF), insulintropin, macrophage colonystimulating factor (M-CSF), plasminogen activators such as alteplase,urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, andteneteplase; nerve growth factor (NGF), osteoprotegerin,platelet-derived growth factor, tissue growth factors, transforminggrowth factor-1, vascular endothelial growth factor, leukemia inhibitingfactor, keratinocyte growth factor (KGF), glial growth factor (GGF), TCell receptors, CD molecules/antigens, tumor necrosis factor (TNF),monocyte chemoattractant protein-1, endothelial growth factors,parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosinalpha 1, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosinbeta 9, thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE)compounds, VLA-4 (very late antigen-4), VLA-4 inhibitors,bisphosponates, respiratory syncytial virus antibody, cystic fibrosistransmembrane regulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable for covalent attachment to a polymer of theinvention include but are not limited to amifostine, amiodarone,aminoglutethimide, amsacrine, anagrelide, anastrozole, asparaginase,anthracyclines, bexarotene, bicalutamide, bleomycin, buserelin,busulfan, cabergoline, capecitabine, carboplatin, carmustine,chlorambucin, cisplatin, cladribine, clodronate, cyclophosphamide,cyproterone, cytarabine, camptothecins, 13-cis retinoic acid, all transretinoic acid; dacarbazine, dactinomycin, daunorubicin, dexamethasone,diclofenac, diethylstilbestrol, docetaxel, doxorubicin, epirubicin,estramustine, etoposide, exemestane, fexofenadine, fludarabine,fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine,epinephrine, L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib,irinotecan, itraconazole, goserelin, letrozole, leucovorin, levamisole,lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, methotrexate, metoclopramide, mitomycin, mitotane,mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate,beclomethasone diprepionate, triamcinolone acetamide, budesonideacetonide, fluticasone, ipratropium bromide, flunisolide, cromolynsodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, non-glycosylated, and biologicallyactive fragments and analogs thereof.

The segmented, water soluble polymers of the invention may employed invarious useful conjugates known in the art; for example, PEG-derivatizedlipids for use in long-circulating liposomes (see e.g. D. Lasic and F.Martin, STEALTH LIPOSOMES, CRC Press, Boca Raton, Fla., 1995). Suchlipids include phospholipids, such as various phosphatidylethanolamines, and are typically attached to the functionalized watersoluble polymer, such as a segmented PEG having an electrophilicfunctional group, via an amine or hydroxyl group on the polar head groupof the phospholipid.

Yet another useful conjugate is a PEG-biotin conjugate. Such a compoundcan serve as a tether in binding of moieties via the well knownnoncovalent biotin-avidin association. A heterobifunctional segmentedpolymer of the invention, having a biotin at one terminus and afunctional group, as described above, at the other terminus, can be usedto tether biotin to any desired molecule or surface having acorresponding functional group; the resulting conjugates can then bebound to avidin-containing molecules or surfaces.

The reactive polymers of the invention may also be attached, eithercovalently or non-covalently, to various solid entities, includingchemical separation and purification surfaces, solid supports forsynthesis, or metal surfaces. One type of functionalized polymer that isespecially useful for attachment to surfaces is silylated PEG, forexample, PEG terminated with a trialkoxysilyl group (see e.g. S. Jo etal., Biomaterials 21(6):605-16, 2000). Such groups form covalentsiloxane bonds to surfaces bearing hydroxyl or oxide groups, such asglass or metals having metal oxide at the surface.

The degree of absorption of cells and proteins to PEG, in comparison toother synthetic materials, is very low (G. Hooftman et al., J. Bioact.Compat. Pol. 11:135, 1996), which is consistent with its lowimmunogenicity. Accordingly, surfaces coated with the water-soluble,biocompatible polymers of the invention can be used, for example, inarterial replacements and other medical and diagnostic devices. Thepolymers of the invention may also be employed in biochemical sensors,bioelectronic switches, and gates.

VII. Pharmaceutical Compositions and Methods of Administration

The invention also provides pharmaceutical preparations comprising aconjugate as provided herein in combination with a pharmaceuticalexcipient. Generally, the conjugate itself will be in solid form, and iscombined with a suitable pharmaceutical excipient that can be in eithersolid or liquid form.

Exemplary excipients include, without limitation, carbohydrates,inorganic salts, antimicrobial agents, antioxidants, surfactants,buffers, acids, bases, and combinations thereof. A carbohydrate such asa sugar, a derivatized sugar such as an alditol, aldonic acid, anesterified sugar, and/or a sugar polymer may be present as an excipient.Specific carbohydrate excipients include, for example: monosaccharides,such as fructose, maltose, galactose, glucose, D-mannose, sorbose, andthe like; disaccharides, such as lactose, sucrose, trehalose,cellobiose, and the like; polysaccharides, such as raffinose,melezitose, maltodextrins, dextrans, starches, and the like; andalditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfate, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both available from BASF, Mount Olive, N.J.);sorbitan esters; lipids, such as phospholipids such as lecithin andother phosphatidyl cholines, phosphatidyl ethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumarate, and combinationsthereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thenfurther exploring the range at which optimal performance is attainedwith no significant adverse effects. Generally, however, the excipientwill be present in the composition in an amount of about 1% to about 99%by weight, preferably from about 5%-98% by weight, more preferably fromabout 15-95% by weight of the excipient, with concentrations less than30% by weight most preferred.

The foregoing pharmaceutical excipients, along with other excipients,are described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., a Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., “Handbook of Pharmaceutical Excipients”, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions encompass various formulations,preferably those suited for injection, e.g., suspensions and solutionsas well as powders that can be reconstituted. The pharmaceuticalcompositions can also take other folins such as syrups, creams,ointments, tablets, powders, and the like. Modes of administrationinclude administration by injection, e.g. parenteral, intravenous,intraarterial, intramuscular, subcutaneous, and intrathecal, as well aspulmonary, rectal, transdermal, transmucosal, and oral delivery.

Suitable formulation types for parenteral administration includeready-for-injection solutions, dry powders for combination with asolvent prior to use, suspensions ready for injection, dry insolublecompositions for combination with a vehicle prior to use, emulsions andliquid concentrates for dilution prior to administration.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition, or prone to acondition, that is responsive to treatment or prevention with theconjugate. The method comprises administering, e.g. via injection, atherapeutically effective amount of the conjugate, preferably providedas part of a pharmaceutical preparation as described above.

The dose to be administered, both unit dosage and dosing schedule, willvary depend upon the age, weight, and general condition of the subject,as well as the severity of the condition being treated, the judgment ofthe health care professional, and the agent being administered.Therapeutically effective amounts are known to those skilled in the artand/or are described in the pertinent reference texts and literature.Generally, a therapeutically effective amount of a conjugate will rangefrom about 0.001 mg to 100 mg, preferably in doses from 0.01 mg/day to75 mg/day, and more preferably in doses from 0.10 mg/day to 50 mg/day.Exemplary dosing schedules include, for example, one to five times aday, one to three times weekly, or one or two times monthly. Once theclinical endpoint has been achieved, dosing of the composition ishalted.

In some embodiments described herein, the segmented polymer conjugatecontains cleavable linkages, which may include the linkage(s) betweenthe POLY_(A) and POLY_(B) segments. This can be advantageous when, forexample, clearance from the body is potentially a problem because of thesize of the conjugate. Optimally, clearance of the conjugate (viacleavage of individual water-soluble polymer portions) can be modulatedby selecting the polymer molecular size and the type and frequency ofdegradable linkage that provides the desired clearance properties. Oneof ordinary skill in the art can determine, using routineexperimentation, a proper molecular size and cleavable functional group,e.g. by first preparing a series of polymers and/or conjugates withdifferent molecular weights and cleavable linkages, and then obtaining aclearance profile by administering the compound to a patient and takingperiodic blood and/or urine samples. Once a clearance profile has beenobtained for each tested conjugate, a suitable conjugate can beidentified.

Examples

The following examples illustrate but are not intended in any way tolimit the invention.

Examples 1 and 2 demonstrate the conversion of the functional group (Y)of a low MW (“POLY_(B)”) polymer from hydroxy to mesylate(methanesulfonate) and p-toluenethiosulfonate, respectively, in goodyield.

Example 1 Synthesis of α-t-Boc amino-ω-methanesulfonate PEG(3400)

BocNH-PEG_(3.4 kD)-OMS

α-N-t-Boc amino-ω-hydroxy PEG(3400) (MW 3318 Da, 4.0 g, 0.0012 mol)(Shearwater Corp.) was azeotroped in chloroform (80 ml) on a rotaryevaporator at 35° C. to dryness, and chloroform (50 ml) was added to theresidual syrup. The solution was cooled to 4° C. under argon, andtriethylamine (0.31 ml, 0.0022 mol) was injected, followed by slowinjection of methanesulfonyl chloride (0.15 ml, 0.0019 mol). Thereaction mixture was stirred overnight under argon while the bath wasallowed to rise to ambient temperature. Anhydrous sodium carbonate (4.0gm) was added to the reaction mixture, and the resulting solution wasstirred at room temperature for one hour. The mixture was then filtered,and the filtrate was concentrated to dryness. Isopropanol (40 ml) wasadded, and the precipitated product was collected by filtration anddried under vacuum to yield 3.7 g α-t-Boc amino-ω-methanesulfonatePEG(3400). NMR (DMSO-d6): 1.37 ppm (s, —OC(CH₃)₃), 3.51 ppm (s, PEGbackbone), 4.31 ppm (t, —CH₂SO₂—), 6.76 ppm (—CH₂NH—CO—).

Example 2 Synthesis of α-t-Boc amino-ω-p-toluenethiosulfonate PEG(3400)

BocNH-PEG_(3.4 kD)-STs

α-t-Boc amino-ω-methanesulfonate PEG(3400) (the product of Example 1)(1.0 g, 0.30 mmol) was azeotroped to dryness in chloroform (30 ml) on arotary evaporator at 35° C., and anhydrous ethanol (15 ml) was added tothe residual syrup. Potassium p-toluenethiosulfonate (292 mg, 1.25 mmol)was added, and the mixture was refluxed under argon overnight. Thesolvent was removed on a rotary evaporator at 40° C., and the residuewas dried under vacuum for 30 minutes. The crude product was dissolvedin 100 ml 1M NaH₂PO₄—Na₂HPO₄ buffer solution (contain 10 wt % NaCl) atpH 5.8, and the resulting solution was extracted with dichloromethane(100 ml×3). The dichloromethane phase was dried over anhydrous sodiumsulfate, filtered, and the filtrate concentrated to near dryness on arotary evaporator. The product was precipitated by addition ofisopropanol/ether (40 ml/20 ml), collected by filtration, and driedunder vacuum. Yield: 0.7 g α-t-Boc amino-ω-p-toluenethiosulfonatePEG(3400). NMR: (DMSO-d6): 1.37 ppm (s, —OC(CH₃)₃), 2.43 ppm (s,CH₃—CH₂═CH₂/Ar), 3.51 ppm (s, PEG backbone), 6.76 ppm (t, —CH₂NH—CO—),7.49 ppm (dd, CH₃—CH₂═CH₂/Ar), 7.82 ppm (dd, CH₃—CH₂═CH₂/Ar).

Example 3 demonstrates the deprotection of the amino group of the lowmolecular weight (“POLY_(B)”) polymer of Example 2 in good yield.

Example 3 Synthesis of α-NH₂-ω-p-toluenethiosulfonate PEG(3400)

H₂N-PEG_(3.4 kD)-STs

α-t-Boc amino-ω-p-toluenethiosulfonate PEG(3400) (the product of Example2) (0.7 g) was dissolved in anhydrous dichloromethane (3.5 ml) andtrifluroacetic acid (3.5 ml) under argon. The solution was stirred atroom temperature for one hour and concentrated to dryness. Isopropanol(20 mL) was added, and the precipitated product was collected byfiltration and dried under vacuum. Yield: 0.6 gα-NH₂-ω-p-toluenethiosulfonate PEG(3400). NMR (DMSO-d6): 2.43 ppm (s,CH₃—CH₂═CH₂/Ar), 2.95 ppm (t, —OCH₂CH₂NH₂), 3.51 ppm (s, PEG backbone),7.49 ppm (dd, CH₃—CH₂═CH₂/Ar), 7.82 ppm (dd, CH₃—CH₂═CH₂/Ar).

Example 4 demonstrates the linking of a high molecular weight(“POLY_(A)”) and low molecular weight (“POLY_(B)”) polymer, by reactionof an amine on POLY_(B) with a benzotriazole carbonate on POLY_(A), toform a functionalized, segmented carbamate-linked polymer. The segmentedpolymer product has a p-toluenethiosulfonate functionality (from thePOLY_(B) segment).

Example 4 Synthesis of mPEG(23.4 kDa)-p-toluenethiosulfonate

mPEG_(20 kD)-O—C(O)—NH-PEG_(3.4 kD)-STs

MPEG(20 kDa)-1-benzotriazole carbonate (813 mg, MW 21 kDa, 0.039 mmol)(Shearwater Corp.) and PEG(3400)-α-NH₂-ω-p-toluenethiosulfonate (theproduct of Example 3) (MW 3805 Da, 200 mg, 0.053 mmol) were dissolved inanhydrous dichloromethane (20 ml) under argon, and triethylamine (30.8μl, 0.22 mmol) was injected. The solution was stirred at roomtemperature overnight, then concentrated to dryness. Isopropanol (10 ml)was added, and the precipitated product was collected by filtration anddried under vacuum. Yield: 843 mg.

The crude mPEG (23.4 kDa)-p-toluenethiosulfonate (560 mg) in 50 mLdeionized water was loaded onto a column packed with 50 ml Poros® media.The column was eluted with 100 ml de-ionized water. Sodium chloride (15g) was added to the eluant, and the resulting solution was extractedwith dichloromethane (100 ml×3). The extract was dried over anhydroussodium sulfate, filtered, and the filtrate concentrated to near drynesson a rotary evaporator. Ethyl ether (50 ml) was added to precipitate theproduct. The product was collected by filtration and dried under vacuum.Yield 495 mg mPEG(23.4 kDa)-p-toluenethiosulfonate. NMR (DMSO-d6): 2.43ppm (s, CH₃—CH₂═CH₂/Ar), 3.51 ppm (s, PEG backbone), 7.23 ppm (t,—NHCOO—), 7.49 ppm (dd, CH₃—CH₂═CH₂/Ar), 7.82 ppm (dd, CH₃—CH₂═CH²/Ar).

Example 5 demonstrates the conjugation of a biologically active agent,α1-antitrypsin, with the segmented polymer derivative of Example 4, viathe functional group p-toluenethiolsulfonate.

Example 5 PEGylation of α1-antitrypsin

mPEG_(20 kD)-O—C(O)—NH-PEG_(3.4 kD)-STs+HS-{α1AT}→mPEG_(20 kD)-O—C(O)—NH-PEG_(3.4 kD)-SS-{α1AT}

To a solution of α1-antitrypsin (1 mg, Sigma, MW 25 kDa) in 100 mMsodium phosphate (pH 7.2, 1 ml) was added 2.8 mg of mPEGp-toluenethiolsulfonate (the product of Example 4) (24 kDa), and thesolution was stirred overnight at room temperature. Capillaryelectrophoresis indicated that the PEG α1-antitrypsin conjugate wasformed in 36% yield. SDS gel electrophoresis also demonstrated thepresence of the PEG conjugate. Treatment of the PEG conjugate withβ-mercaptoethanol resulted in the formation of α1-antitrypsin, asevidenced by gel electrophoresis, thus indicating the presence of adisulfide linkage in the PEG α1-antitrypsin conjugate.

Example 6 provides another illustration of the linking of a highmolecular weight (“POLY_(A)”) and low molecular weight (“POLY_(B)”)polymer, by reaction of an amine on POLY_(B) with a benzotriazolecarbonate on POLY_(A), to form a functionalized, segmentedcarbamate-linked polymer. In this case, the segmented polymer producthas a carboxylic acid functionality (from the POLY_(B) segment).

Example 6 mPEG(22 KDa)-propionic acid

mPEG_(20 kD)-O—C(O)-Bt+H₂N-PEG_(2 kD)-CH₂CH₂C(O)OH→mPEG_(20 kD)-O—C(O)—NH-PEG_(2 kD)-CH₂CH₂C(O)OH

To a solution of mPEG(20 KDa)-benzotriazole carbonate (2.0 g, 0.1 mmol)(Shearwater Corporation) in methylene chloride (20 ml) were added PEG(2KDa)-α-amino-ω-propionic acid (0.24 g, 0.12 mmol) (ShearwaterCorporation) and triethylamine (0.06 ml), and the reaction mixture wasstirred overnight at room temperature under argon. The mixture wasfiltered and evaporated to dryness. The crude product was dissolved inmethylene chloride and precipitated with isopropyl alcohol, and theprecipitate was dried under reduced pressure. Yield 1.9 g. NMR(d₆-DMSO): 2.44 ppm (t, —CH₂—COO—), 3.11 ppm (q, —CH _(2—)NH—), 3.24 ppm(s, —OCH₃), 3.51 ppm (s, PEG backbone), 4.04 ppm (m, —CH_(2—)O(C═O)—),7.11 ppm (t, —(C═O)—NH—). Anion exchange chromatography yielded mPEG(22KDa)-propionic Acid (93%) and mPEG-20 KDa (7%).

Example 7 demonstrates the conversion of the functional group, Y, of thesegmented polymer produced in Example 6 from propionic acid to propionicacid N-hydroxysuccinimide ester.

Example 7 mPEG(22 KDa)-propionic acid N-hydroxysuccinimide ester

mPEG_(20 kD)-O—C(O)—NH-PEG_(2 kD)-CH₂CH₂C(O)O-succinimide

To a solution of mPEG(22 KDa)-propionic acid (the product of Example 6)(1.1 g, 0.050 mmol) in anhydrous methylene chloride (10 ml),N-hydroxysuccinimide (0.0063 g, 0.055 mmol) was added, followed by1,3-dicyclohexylcarbodiimide (1.0 M solution in methylene chloride, 0.05ml, 0.055 mmol). The reaction mixture was stirred overnight at roomtemperature under argon. The mixture was filtered and the solvent wasevaporated. The crude product was dissolved in methylene chloride,precipitated with isopropyl alcohol, and dried under reduced pressure.Yield 0.9 g. NMR (d₆-DMSO): 2.81 ppm (s, —CH₂—CH₂— (succinate)), 2.92ppm (t, —CH₂—COO—), 3.11 ppm (q, —CH ₂—NH—), 3.24 ppm (s, —OCH₃), 3.51ppm (s, PEG backbone), 4.03 ppm (m, —CH₂—O(C═O)—), 7.11 ppm (t,—(C═O)—NH—).

Example 8 demonstrates the conversion of the functional group (Y) of alow MW (“POLY_(B)”) polymer from propionic acid to methyl propionate.

Example 8 PEG(2 KDa)-α-amino-ω-propionic acid methyl ester

H₂N-PEG₂₀₀₀-CH₂CH₂C(O)OCH₃

To a solution of PEG(2 KDa)-α-amino-ω-propionic acid (10 g, 0.0050 mol)(Shearwater Corporation) in anhydrous methylene chloride (100 ml),1-hydroxybenzotriazole (0.30 g), 4-(dimethylamino)pyridine (1.0 g),methanol (3.2 g, 0.100 mol) and 1,3-dicyclohexylcarbodiimide (1.0 Msolution in methylene chloride, 7.5 ml, 0.0075 mol) were added. Thereaction mixture was stirred overnight at room temperature under argon.The mixture was concentrated to about 50 ml, filtered, and added to 800ml of cold diethyl ether. The precipitated product was filtered anddried under reduced pressure. Yield 9.5 g. NMR (d₆-DMSO): 2.53 ppm (t,—CH₂—COO—), 2.95 ppm (t, —CH₂-amine), 3.51 ppm (s, PEG backbone).

Example 9 illustrates the linking of a high molecular weight(“POLY_(A)”) and low molecular weight (“POLY_(B)”) polymer, by reactionof an amine on POLY_(B) with a benzotriazole carbonate on POLY_(A), toform a functionalized, segmented carbamate-linked polymer. In this case,the segmented polymer product has a carboxylic ester functionality (fromthe POLY_(B) segment).

Example 9 mPEG(32 KDa)-propionic acid methyl ester

mPEG_(300 kD)O—C(O)-Bt+H₂N-PEG₂₀₀₀-CH₂CH₂C(O)OCH₃→mPEG_(300 kD)-O—C(O)—NH-PEG₂₆₀₀-CH₂CH₂C(O)OCH₃

To a solution of mPEG(30 KDa)-benzotriazole carbonate (3.0 g, 0.1 mmol)(Shearwater Corporation) in methylene chloride (20 ml), PEG(2KDa)-α-amino-ω-propionic acid methyl ester (the product of Example 8)(0.24 g, 0.12 mmol) and triethylamine (0.060 ml) were added, and thereaction mixture was stirred overnight at room temperature under argon.The mixture was filtered and the solvent was evaporated. The crudeproduct was dissolved in methylene chloride, precipitated with isopropylalcohol, and dried under reduced pressure. Yield 2.8 g. NMR (d₆-DMSO):2.53 ppm (t, —CH₂—COO—), 3.11 ppm (q, —CH ₂NH—), 3.24 ppm (s, —OCH₃),3.51 ppm (s, PEG backbone), 4.04 ppm (m, —CH_(2—)O(C═O)—), 7.11 ppm (t,—(C═O)—NH—).

Examples 10 and 11 demonstrate the conversion of the functional group,Y, of the segmented polymer produced in Example 9 from methyl propionateto propionic acid and propionic acid NHS ester, respectively.

Example 10 mPEG(32 KDa)-propionic acid

mPEG_(30 kD)-O—C(O)—NH-PEG₂₀₀₀-CH₂CH₂C(O)OH

mPEG(32 KDa)-propionic acid methyl ester (the product of Example 9) (2.8g, 0.082 mmol) was dissolved in 20 ml deionized water, and the pH wasadjusted to 12.0 with 0.5 M NaOH solution. The reaction mixture wasstirred 1.5 h at pH 12.0. Sodium chloride (3 g) was added, and the pHwas adjusted to 3 with 5% phosphoric acid. The product was extractedwith methylene chloride 3 times, and the combined extracts were driedwith anhydrous magnesium chloride. The solvent was removed and theproduct dried under reduced pressure. Yield 1.6 g. NMR (d₆-DMSO): 2.44ppm (t, —CH₂—COO—), 3.11 ppm (q, —CH ₂—NH—), 3.24 ppm (s, —OCH₃), 3.51ppm (s, PEG backbone), 4.04 ppm (m, —CH_(2—)O(C═O)—), 7.11 ppm (t,—(C═O)—NH—).

Anion exchange chromatography provided mPEG(32 KDa)-propionic acid(97.5%), Mpeg-30 KDa (2.5%).

Example 11 mPEG(32 KDa)-propionic acid, N-hydroxysuccinimide ester

MPEG_(30 kD)-O—C(O)—NH-PEG₂₀₀₀-CH₂CH₂C(O)O-succinimide

To a solution of mPEG(32 KDa) propionic acid (product of Example 10)(1.6 g, 0.050 mmol) in anhydrous methylene chloride (10 ml),N-hydroxysuccinimide (0.0063 g, 0.055 mmol) was added, followed by1,3-dicyclohexylcarbodiimide (1.0 M solution in methylene chloride, 0.05ml, 0.055 mmol). The reaction mixture was stirred overnight at roomtemperature under argon, filtered, and concentrated. The crude productwas dissolved in methylene chloride, precipitated with isopropylalcohol, and dried under reduced pressure. Yield 0.9 g. NMR (d₆-DMSO):2.81 ppm (s, —CH₂—CH₂— (succinate)), 2.92 ppm (t, —CH₂—COO—), 3.11 ppm(q, —CH ₂—NH—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone), 4.03ppm (m, —CH₂—O(C═O)—), 7.11 ppm (t, —(C═O)—NH—).

Example 12 illustrates the linking of a high molecular weight(“POLY_(A)”) and low molecular weight (“POLY_(B)”) polymer, by reactionof an amine on POLY_(B) with a benzotriazole carbonate on POLY_(A), toform a functionalized, segmented carbamate-linked polymer. In this case,the segmented polymer product has a butanoic acid functionality (fromthe POLY_(B) segment).

Example 12 mPEG(23.4 KDa)-butanoic acid

mPEG_(20 kD)-O—C(O)-Bt+H₂N-PEG_(3.4 kD)-CH₂CH₂CH₂C(O)OH→mPEG_(20 kD)-O—C(O)—NH-PEG_(3.4 kD)-CH₂CH₂CH₂C(O)OH

To a solution of mPEG(20 KDa)-benzotriazole carbonate (2.0 g, 0.1 mmol)(Shearwater Corporation) in methylene chloride (20 ml), PEG(3.4KDa)-α-amino-ω-butanoic acid (0.45 g, 0.12 mmol) (ShearwaterCorporation) and triethylamine (0.060 ml) were added, and the reactionmixture was stirred overnight at room temperature under argonatmosphere. The mixture was filtered and evaporated to dryness. Thecrude product was dissolved in methylene chloride, precipitated withisopropyl alcohol, and dried under reduced pressure. Yield 2.2 g. NMR(d₆-DMSO): 1.72 ppm (q, CH ₂—CH₂—COO—) 2.24 ppm (t, —CH₂—COO—), 3.11 ppm(q, —CH ₂—NH—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone), 4.04ppm (m, —CH₂—O(C═O)—), 7.11 ppm (t, —(C═O)—NH—). Anion exchangechromatography yielded mPEG(23.4 KDa)-butanoic acid (92%), MPEG-20 KDa(8%).

Example 13 demonstrates the modification of the functional group, Y, ofthe segmented polymer produced in Example 12 from butanoic acid tobutanoic acid N-hydroxysuccinimide ester.

Example 13 mPEG(22 KDa)-butanoic acid, N-hydroxysuccinimide ester

MPEG_(20 kD)O—C(O)—NH-PEG_(3.4 kD)-CH₂CH₂CH₂C(O)O-succinimide

To a solution of mPEG(23.4 KDa)-butanoic acid (product of Example 12)(1.17 g, 0.050 mmol) in anhydrous methylene chloride (10 ml),N-hydroxysuccinimide (6.3 mg, 0.055 mmol) was added, followed by1,3-dicyclohexylcarbodiimide (1.0 M solution in methylene chloride, 0.05ml, 0.055 mmol). The reaction mixture was stirred overnight at roomtemperature under argon, filtered and evaporated to dryness. The crudeproduct was dissolved in methylene chloride, precipitated with isopropylalcohol, and dried under reduced pressure. Yield 1.0 g. NMR (d₆-DMSO):1.83 ppm (q, CH ₂—CH₂—COO—), 2.70 ppm (t, —CH₂—COO—), 2.81 ppm (s,—CH₂—CH₂— (succinate)), 2.92 ppm, 3.11 ppm (q, —CH ₂—NH—), 3.24 ppm (s,—OCH₃), 3.51 ppm (s, PEG backbone), 4.03 ppm (m, —CH₂—O(C═O)—), 7.11 ppm(t, —(C═O)—NH—).

Examples 14A-C illustrate the linking of a medium- or high-molecularweight (“POLY_(A)”) polymer and low molecular weight (“POLY_(B)”)polymer (triethylene glycol diamine), by reaction of an amine onPOLY_(B) with a succinimide ester or benzotriazole carbonate onPOLY_(A), to form a functionalized, segmented carbamate-linked polymer.In this case, the segmented polymer product has an amine functionality(from the POLY_(B) segment). Examples 14A and 14C illustrate preparationon a 200 g scale.

Example 14A mPEG(2 KDa) amine

mPEG_(20 kD)-O—C(O)—O—SC+H₂N—(CH₂CH₂O)₂—CH₂CH₂—NH₂→mPEG_(20 kD)-O—C(O)—NH—(CH₂CH₂O)₂—CH₂CH₂—NH₂

To a solution of 200 g (0.1 eq) of mPEG₂₀₀₀ in 1 L acetonitrile wasadded 43.55 g (0.17 eq) disuccinimidyl carbonate. Upon dissolution, 24.4ml anhydrous pyridine was added dropwise via syringe. The solution wasthen allowed to stir for 12 hours, filtered under argon, andconcentrated to remove solvent. The residue was redissolved in 2 Ldichloromethane, and the solution was washed with two 400 mL portions of10% NaH₂PO_(4—: 25)% NaCl. The aqueous extracts were back-extracted with400 mL dichloromethane. The organic phase was combined, dried oversodium sulfate, filtered, and concentrated. This residue was taken up in5 L of ether and stirred in a warm water bath under argon. Uponclarification the mixture was cooled in an ice bath, and thecrystallized solid was filtered and dried under vacuum.

This product (mPEG₂₀₀₀-succinimide) (206 g) was dissolved in 4.12 Ldichloromethane. The solution was added dropwise with vigorous stirring,under argon, to a solution of 300 mL 2,2′-(ethylenedioxy)bis(ethylamine)(H₂NCH₂CH₂OCH₂CH₂OCH₂CH₂NH₂). The resulting solution was stirred for 12h under argon, then concentrated under reduced pressure to give aviscous oil. This residue was taken up in a minimum amount ofdichloromethane and precipitated by addition of 2 L isopropylalcohol/2.5 L ether. The precipitated solid was filtered and dried undervacuum.

Example 14B mPEG(20 KDa) amine

mPEG_(200 kD)-O—C(O)—O-Bt+H₂N—(CH₂CH₂O)₂—CH₂CH₂—NH₂→mPEG_(200 kD)-O—C(O)—NH—(CH₂CH₂O)₂—CH₂CH₂—NH₂

To a solution of mPEG(20 KDa)-benzotriazole carbonate (2.0 g, 0.1 mmol)(Shearwater Corporation) in methylene chloride (20 ml),2,2′-(ethylenedioxy)bis(ethylamine) (MW 148.21, 0.3 g, 2.0 mmol) wasadded, and the reaction mixture was stirred 2 h at room temperatureunder argon. The solvent was evaporated to dryness, and the crudeproduct was dissolved in methylene chloride and precipitated withisopropyl alcohol. The product was dried under reduced pressure. Yield1.8 g. NMR (d₆-DMSO): 2.64 ppm (t, —CH₂-amine-), 3.11 ppm (q, —CH₂—NH—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone), 4.04 ppm (m,—CH₂—O(C═O)—), 7.11 ppm (t, —(C═O)—NH—). Cation exchange chromatographyyielded mPEG(20K)-amine (97.5%).

Example 14C mPEG(20 KDa) amine

mPEG_(200 kD)-O—C(O)—O-Bt+H₂N—(CH₂CH₂O)₂—CH₂CH₂—NH₂→mPEG_(200 kD)-O—C(O)—NH—(CH₂CH₂O)₂—CH₂CH₂—NH₂

To 29.2 mL of 2,2-(ethylenedioxy)bis(ethylamine) was added, dropwiseunder argon via cannula, with vigorous stirring, a solution of 199.2 gmPEG(20 KDa)-benzotriazole carbonate and 0.4 g BHT in 4 Ldichloromethane. The reaction mixture was allowed to stir overnight, anda sample was withdrawn to check the completeness of the reaction. Themixture was concentrated under reduced pressure, and the residue wastaken up in a minimum amount of dichloromethane. The product wasprecipitated by adding 4 L of 1:1 isopropyl alcohol/ether and cooling inan ice bath. The product was filtered and washed with 3 L of ether toremove any excess reagent.

Example 15 demonstrates the conversion of the functional group of thelow weight polymer, PEG(3.4 KDa)-α-hydroxy-ω-propionaldehyde, from ahydroxy to an amine group.

Example 15 PEG(3.4 KDa)-α-amine-ω-propionaldehyde diethyl acetal

H₂N-PEG_(3.4 kD)-CH₂CH₂CH(OCH₂CH₃)₂

To a solution of PEG(3.4 KDa)-α-hydroxy-ω-propionaldehyde diethyl acetal(NOF Corporation, Tokyo, JP) (1.0 g, 0.294 mmol) in a mixture of toluene(20 ml) and dichloromethane (5 ml), triethylamine (0.07 ml, 0.502 mmol,171% of stoichiometric amount) and methanesulfonyl chloride (0.028 ml,0.362 mmol, 123% of stoichiometric amount) were added, and the resultingmixture was stirred overnight under nitrogen atmosphere. The mixture wasfiltered, and the solvent was distilled off under reduced pressure. Theresidue was added to a mixture of 16 ml concentrated NH₄OH and 1.6 gNH₄Cl and stirred 42 hours at room temperature. The reaction product wasextracted with dichloromethane (3 times 20 ml). The extract was washedwith 5 ml 1M HCl and 5 ml distilled water and dried with anhydroussodium sulfate. The solvent was distilled under reduced pressure, giving0.78 g of PEG(3.4 KDa)-α-amine hydrochloride-wpropionaldehyde diethylacetal. NMR (d₆-DMSO): 1.10 ppm (t, CH₃—, acetal), 1.74 ppm (q, —OCH₂ CH₂CH—, acetal), 2.94 ppm (t, —CH₂-amine hydrochloride), 3.51 ppm (s, PEGbackbone), 4.55 ppm (t, —CH—, acetal), 7.11 ppm (t, —(C═O)—NH—).

Example 16 demonstrates the linking of the low molecular weight(“POLY_(B)”) polymer produced in Example 15, PEG(3.4KDa)-α-amine-ω-propionaldehyde diethyl acetal, with a high molecularweight (“POLY_(B)”) polymer, by reaction of an amine on POLY_(B) with abenzotriazole carbonate on POLY_(A), to form a functionalized, segmentedcarbamate-linked polymer. In this case, the segmented polymer producthas a propionaldehyde diethyl acetal functionality (from the POLY_(B)segment).

Example 16 mPEG(23.4 KDa)-propionaldehyde, diethyl acetal

mPEG_(20 kD)O—C(O)-Bt+H₂N-PEG_(3.4 kD)-CH₂CH₂CH(OCH₂CH₃)₂→mPEG_(20 kD)-O—C(O)—NH-PEG_(3.4 kD)-CH₂CH₂CH(OCH₂CH₃)₂

To a solution of mPEG(20 KDa)-benzotriazole carbonate (2.0 g, 0.1 mmol)(Shearwater Corporation) in methylene chloride (20 ml), PEG(3.4KDa)-α-amine-ω-propionaldehyde diethyl acetal, the product of Example 15(0.36 g, 0.106 mmol) was added, and the reaction mixture was stirredovernight at room temperature under argon atmosphere. The solvent wasevaporated to dryness, and the crude product was dissolved in methylenechloride and precipitated with isopropyl alcohol. The precipitate wasdried under reduced pressure. Yield 1.8 g. NMR (d₆-DMSO): 1.10 ppm (t,CH₃—, acetal), 1.74 ppm (q, —OCH₂ CH ₂CH—, acetal), 3.11 ppm (q, —CH₂—NH—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone), 4.04 ppm (m,—CH₂—O(C═O)—), 4.55 ppm (t, —CH—, acetal), 7.11 ppm (t, —(C═O)—NH—).

Example 17 demonstrates the conversion of the functional group, Y, ofthe segmented polymer produced in Example 16 from propionaldehydediethyl acetal to propionaldehyde.

Example 17 mPEG(23.4 KDa)-propionaldehyde

mPEG_(20 kD)-O—C(O)—NH-PEG_(3.4 kD)-CH₂CH₂CHO

mPEG(23.4 KDa)-propionaldehyde, diethyl acetal (product of Example 16)(1.8 g) was dissolved in 20 ml water, and the pH of the solution wasadjusted to 3 with dilute phosphoric acid. The solution was stirred for3 hours at room temperature, and 0.5M sodium hydroxide was added toadjust the pH of the solution to 7. The product was extracted withmethylene chloride, the extract was dried with anhydrous magnesiumsulfate, and the solvent was distilled off under reduced pressure.Yield: 1.6 g. NMR (d₆-DMSO): 2.60 ppm (dt, —OCH₂ CH₂ CH—, aldehyde),3.24 ppm (q, —CH ₂—NH—), 3.51 ppm (s, PEG backbone), 4.04 ppm (m,—CH₂—O(C═O)—), 7.11 ppm (t, —(C═O)—NH—), 9.65 ppm (t, —CH, aldehyde).

Example 18 demonstrates the linking of the low molecular weight(“POLY_(B)”) polymer produced in Example 15 with a high molecular weight(“POLY_(B)”) branched polymer, by reaction of an amine on POLY_(B) withan NHS ester on POLY_(A), to form a functionalized, segmentedamide-linked polymer. The segmented polymer product has apropionaldehyde diethyl acetal functionality (from the POLY_(B)segment).

Example 18 Branched PEG2(43.4 KDa)-propionaldehyde, diethyl acetal

To a solution of branched PEG2 (40 KDa)-N-hydroxysuccinimide ester (1.0g, 0.025 mmol) (Shearwater Corporation) in methylene chloride (8 ml),PEG_(3.4 kDa)-α-amine hydrochloride-ω-propionaldehyde diethyl acetal(the product of Example 15) (0.12 g, 0.035 mmol) and triethylamine (0.01ml) were added, and the reaction mixture was stirred overnight at roomtemperature under argon. The solvent was evaporated to dryness, and thecrude product was dissolved in methylene chloride and precipitated withdiethyl ether. The precipitate was dried under reduced pressure. Yield0.83 g. NMR (d₆-DMSO):1.10 ppm (t, CH₃—, acetal), 1.74 ppm (q, —OCH₂ CH₂CH—, acetal), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone), 4.55 ppm(t, —CH—, acetal).

Example 19 demonstrates the conversion of the endgroup, Y, of thesegmented polymer produced in Example 18 from propionaldehyde diethylacetal to propionaldehyde.

Example 19 Branched PEG2(43.4 KDa)-propionaldehyde

Branched PEG2(43.4 KDa)-propionaldehyde diethyl acetal (the product ofExample 18) (0.4 g) was dissolved in 10 ml water, and the pH of thesolution was adjusted to 3 with dilute phosphoric acid. The solution wasstirred for 3 hours at room temperature, and 0.5M NaOH was added toadjust the pH of the solution to 7. The product was extracted withmethylene chloride. The extract was dried with anhydrous magnesiumsulfate and the solvent distilled off under reduced pressure. Yield 0.35g. NMR (d₆-DMSO): 2.60 ppm (dt, —OCH₂ CH₂ CH—, aldehyde), 3.24 ppm (s,—OCH₃), 3.51 ppm (s, PEG backbone), 9.65 ppm (t, —CH, aldehyde).

Example 20 demonstrates the linking of a low molecular weight(“POLY_(B)”) polymer with a high molecular weight (“POLY_(B)”) polymer,by reaction of an amine on POLY_(B) with a benzotriazole carbonate onPOLY_(A), to form a functionalized, segmented carbamate-linked polymer.In this case, the segmented polymer product has a maleimidefunctionality (from the POLY_(B) segment).

Example 20 MPEG_(20K)-Maleimide

mPEG_(20 kD)-O—C(O)-Bt+TFA⁻H₃N⁺—(CH₂CH₂O)₂—CH₂CH₂-maleimide→mPEG_(20 kD)-O—C(O)—NH—(CH₂CH₂O)₂—CH₂CH₂-maleimide

To a solution of mPEG(20 KDa)-benzotriazole carbonate (20.0 g, 0.001mol) (Shearwater Corporation) in methylene chloride (200 ml),maleimide-triethyleneglycol-amine TFA (0.68 g, 0.002 mol) and4-methylmorpholine (0.44 ml, 0.004 mol) were added. The reaction wasstirred 4 hours at room temperature under argon. The solvent wasevaporated to dryness and the product precipitated with isopropylalcohol (1000 ml). The precipitate was collected by vacuum filtrationand dried in vacuo overnight. Yield: 19.5 g. NMR (d6-DMSO): 3.11 ppm(q,—CH₂ —NH—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone), 4.04 ppm(t,—CH₂—O(C═O)—), 7.04 (s, —(C═O)—CH═CH—(C═O)—), 7.11 ppm (t,—(C═O)—NH—).

1. A water soluble polymer having a structure encompassed within thefollowing formula:

wherein: each PEG is a poly(ethylene glycol) segment having a molecularweight ranged of from about 15000 to about 50000; X is —NH—; POLY_(B) isa water soluble segment comprising 1 to about 120 monomeric units, whichhas a lower molecular weight than that of each PEG; [Spacer Group] is agroup linking Y to a terminus of POLY_(B); and Y is functional group orprotected form thereof.
 2. The polymer of claim 1, wherein POLY_(B)comprises one monomer or up to three different monomers selected fromthe group consisting of alkylene glycol, olefinic alcohol,vinylpyrrolidone, hydroxyalkylmethacrylamide, hydroxyalkylmethacrylate,saccharide, α-hydroxy acid, phosphazene, oxazoline, andN-acryloylmorpholine.
 3. The polymer of claim 1, wherein the molecularweight of each PEG is at least twice that of POLY_(B).
 4. The polymer ofclaim 1, wherein POLY_(B) comprises at least 2 monomeric units.
 5. Thepolymer of claim 1, wherein POLY_(B) is a poly(alkylene glycol) segment.6. The polymer of claim 5, wherein POLY_(B) is a poly(ethylene glycol)segment.
 7. The polymer of claim 1, wherein POLY_(B) is poly(ethyleneglycol) segment having a molecular weight selected from the groupconsisting of about 88, about 132, about 176 and about
 220. 8. Thepolymer of claim 1, wherein the Spacer Group is a hydrolytically stablegroup.
 9. The polymer of claim 1, wherein the Spacer Group is ahydrocarbon spacer.
 10. The polymer of claim 1, wherein the hydrocarbonspacer is a linear divalent alkyl chain.
 11. The polymer of claim 1,wherein the linear divalent alkyl chain has 4 to about 8 carbons. 12.The polymer of claim 1, wherein Y is a protected functional group. 13.The polymer of claim 1, wherein Y is a functional group.
 14. The polymerof claim 13, wherein the functional group is a nucleophilic group. 15.The polymer of claim 14, wherein the nucleophilic group is selected fromthe group consisting of hydroxyl, amine, hydrazine, hydrazide and thiol.16. The polymer of claim 13, wherein the functional group is anelectrophilic group.
 17. The polymer of claim 16, wherein theelectrophilic group is selected from the group consisting of carboxylicacid, carboxylic ester, imide ester, orthoester, carbonate, isocyanate,isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, halosilane andphosphoramidate.
 18. The polymer of claim 17, wherein the electrophilicgroup is a carboxylic acid.
 19. The polymer of claim 17, wherein theelectrophilic group is a carboxylic ester.
 20. The polymer of claim 17,wherein the electrophilic group is an aldehyde.
 21. The polymer of claim17, wherein the electrophilic group is a maleimide.
 22. The polymer ofclaim 1, wherein the each PEG is end-capped with a capping groupselected from the group consisting of C₁-C₂₀ alkoxy, C₁-C₂₀ aryloxy anda phospholipid.
 23. The polymer of claim 22, wherein the capping groupis C₁-C₅ alkoxy.
 24. The polymer of claim 23, wherein the capping groupis methoxy.
 25. The polymer of claim 1, conjugated to a biologicallyactive molecule.